Speaking from the White House, Obama said any decisions on the future of Crimea, a pro-Russian area of Ukraine, must include the country's new government.
"The proposed referendum on the future of Crimea would violate the constitution and violate international law," Obama said. "We are well beyond the days when borders can be redrawn over the heads of democratic leaders."
Russian President Vladimir Putin was almost certainly behind Thursday's dramatic developments, but it was not clear whether he is aiming for outright annexation, or simply strengthening his hand in talks with the West.
The U.S. moved to impose financial sanctions and travel restrictions on opponents of Ukraine's new government and the EU also announced limited punitive measures against Putin's government, including the suspension of trade and visa talks. Both Washington and the EU said they were discussing further sanctions.
"I am confident that we are moving forward together, united in our determination to oppose actions that violate international law and to support the government and people of Ukraine," Obama said.
Crimea's parliament rammed through what amounted to a declaration of independence from Ukraine, announcing it would let the Crimean people, 60 percent of whom are ethnic Russian, decide in a March 16 referedum whether they want to become part of their gigantic neighbor to the east.
"This is our response to the disorder and lawlessness in Kiev," said lawmaker Sergei Shuvainikov. "We will decide our future ourselves."
Ukraine's prime minister swiftly denounced the action. "This so-called referendum has no legal grounds at all," said Arseniy Yatsenyuk. The country's acting president, Oleksandr Turchynov, later said Ukraine would move to dissolve Crimea's parliament, but such an action would have virtually no practical effect.
In Washington, Obama signed an executive order authorizing the Treasury Department to levy financial sanctions against "individuals and entities" deemed responsible for Russia's military takeover in Crimea. The U.S. also imposed a separate ban on U.S. visas for an unspecified and unidentified number of people the U.S. accuses of threatening Ukraine's sovereignty and territorial borders.
In a statement, the White House said the penalties would target "those who are most directly involved in destabilizing Ukraine, including the military intervention in Crimea, and does not preclude further steps should the situation deteriorate."
The U.S. actions came as EU leaders gathered at an emergency summit in Brussels to put in place their own measures, but appeared split over how forcefully to follow America's lead. EU President Herman Van Rompuy said the bloc would suspend talks with Russia on a wide-ranging economic pact and on a visa deal, and would consider further measures if Russia does not quickly open meaningful dialogue.
The Europeans were divided between nations close to Russia's borders, which want the bloc to stand up to Moscow, and some Western economic powerhouses — notably Germany — that were taking a more dovish line.
"Not everyone will be satisfied with the decision, but I should say that we did much more together than one could have expected several hours ago," said Polish Prime Minister Donald Tusk.
Meanwhile, in Moscow, a prominent member of Russia's parliament, Sergei Mironov, said he had introduced a bill to simplify the procedure for Crimea to join Russia and it could be passed as soon as next week. Another senior lawmaker, Leonid Slutsky, said the parliament could consider such a motion after the referendum.
Earlier this week, Putin said Russia had no intention of annexing Crimea, while insisting its population has the right to determine the region's status in a referendum. A popular vote would give Putin a democratic fig leaf for what would effectively be a formal takeover — although it was too early to tell whether such a move would actually go forward.
For Putin, Crimea would be a dazzling acquisition, and help cement his authority with a Russian citizenry that has in recent years shown signs of restiveness and still resents the loss of the sprawling empire Moscow ruled in Soviet times. The peninsula was once Russia's imperial crown jewel, a lush land seized by Catherine the Great in the 18th century that evokes Russia's claim to greatness as a world power.
A referendum had previously been scheduled in Crimea for March 30, but the question to be put to voters was whether their region should enjoy "state autonomy" within Ukraine.
The city legislature in Sevastopol, the Crimean port that hosts Russia's naval base, voted late Thursday to join the referendum. The vote was necessary because the city has an autonomous status making it separate from the rest of Crimea.
Crimea's new leader has said pro-Russian forces numbering more than 11,000 now control all access to the peninsula in the Black Sea and have blockaded all military bases that have not yet surrendered.
EU economic sanctions against Russia could prove painful for Europe since Russia could hit back by turning off the taps to natural gas that is an urgent need for many European countries, including regional giant Germany.
The fallout for Europe from any action targeting influential Russian oligarchs or corporations would also be great. Russian investors hold assets worth billions in European banks, particularly in Britain — which is reluctant to undermine its massive financial services industry. Russia, the EU's third biggest trading partner, bought $170 billion in European machinery, cars and other exports in 2012.
In Simferopol, Crimea's capital, about 50 people rallied outside the local parliament Thursday waving Russian and Crimean flags. "Russia, defend us from genocide," one poster read.
"Only Russia can give us a peaceful life," declared 35-year-old Igor Urbansky, one of the rally particants.
"We are tired of revolutions, maidans and conflicts and we want to live peacefully in Russia," he said, evoking the name of the downtown square in Kiev where tens of thousands of protesters contested the rule of Ukrainian President Viktor Yanukovych, who fled to Russia.
Still not all in the city favored the lawmakers' vote to secede from Ukraine.
"This is crazy. Crimea has become Putin's puppet," said Viktor Gordiyenko, 46. "A referendum at gunpoint of Russia weapons is just a decoration for Putin's show. A decision on occupation has already been made."
Svetlana Savchenko, a Crimean lawmaker, said the choice she and her fellow deputies took in favor of joining Russia will force Moscow to make a decision.
"Now the Russian Federation must begin a procedure — will it take us in or not?" she said.
Rustam Temirgaliev, first vice premier of the Crimean government, said preparations were already underway already to bring Crimea into Russia's "ruble zone."
"At the present moment, a large, important group of specialists from Russia is at work, preparing to assure the entry of Crimea into the Russian Federation," Temirgaliev said.
At the Ukrainian naval base in Novo-Ozerne, the inlet leading to the Black Sea was blocked Thursday by a partially submerged Russian naval vessel, preventing two Ukrainian ships from leaving port. Ukrainian sailors said the Russians had blown up the decommissioned vessel overnight.
Meanwhile, turmoil brewed elsewhere in the Russian-dominated eastern half of Ukraine.
Clashes between protesters and police broke out in the ethnic Russian stronghold of Donetsk as police cleared demonstrators from the regional administration center. The Ukrainian flag was once again hoisted over the building, and about 100 Ukrainian troops could be seen in and around it.