Parliamentarians passed a resolution of no confidence in the government of Prime Minister Yury Yekhanurov with 250 votes in favour, well over the 226 needed for it to pass.
"We should defend the interests of Ukraine. We should defend the Ukrainian people," said Nestor Shufrich, one of the deputies opposed to last week's deal ending a "gas war" with Russian energy giant Gazprom.
While the resolution takes immediate effect, the parliamentarians also ordered that ministers stay on in a caretaker capacity until a new government can be formed.
Analysts were doubtful that would happen before parliamentary elections that are scheduled for March 26.
But the vote is a blow to the leadership of President Viktor Yushchenko.
The cabinet had been expected to weather a torrent of criticism of last week's gas deal, which resolved a crisis that briefly disrupted supplies to several European countries for which Ukraine is the main transit route.
In the event, the no-confidence resolution drew support from diverse political groups.
Votes in favour came from blocs led both by Yulia Tymoshenko, a key figure who stood side-by-side with Yushchenko in the 2004 "orange revolution", and from Viktor Yanukovich, whose pro-Moscow leadership the revolution was aimed at.
Arriving on a visit to the Kazakh capital Astana, Mr Yushchenko slammed the move and said he would challenge it before the Constitutional Court.
"Time will show that this decision is not constitutional," said Mr Yushchenko, who was scheduled to attend the inauguration of Kazakh President Nursultan Nazarbayev.
Mr Yushchenko has been struggling to live up to the hopes raised by the 2004 "orange" protests at which he promised a radical shift away from Moscow and towards the West.
His team last year made heavy work of promised liberalisation, while
Mr Yushchenko also fell out with the charismatic Mrs Tymoshenko, whom he sacked as prime minister in September.
His goals of hoped-for European Union and NATO membership will hang in the balance at the March 26 polls, analysts say.
At Tuesday's session, Mr Yekhanurov defended the deal with Russia on gas prices, telling parliament that it served Ukraine's interests and promising that consumers would not face price hikes any time soon.
"In its actions, the government was guided and will continue to be guided by the national interests of Ukraine," Mr Yekhanurov said.
Ukraine will pay a "far lower" price for gas than other eastern European countries, he said, adding that "gas prices for the population will remain at their current level" for the time being.
European politicians breathed a sigh of relief when Ukraine clinched last week's agreement.
Under the terms of the accord, Ukraine agreed to buy natural gas from both Russia and Turkmenistan through an intermediary company at a rate of US$95 per 1,000 cubic meters.
Thanks to the low prices of supplies from Turkmenistan that is well below the US$230 Gazprom has demanded for Russian gas.
But opponents were angered that the deal still represented a substantial rise on last year's price of US$50 per 1,000 cubic metres.
Some criticised the fact that all supplies to Ukraine will be handled by a single intermediary, RosUkrEnergo, a commercial enterprise owned by Gazprom and an Austrian bank.
The parliamentary vote is likely to weaken Mr Yushchenko, although it is unlikely that the ambitious Mrs Tymoshenko will manage to form a credible government of her own, said political analyst Oleksander Litvinenko of the Razumkov Centre.
"It is doubtful that Mrs Tymoshenko can find a common language" with old "orange revolution" adversaries, Mr Litvinenko said.
Nonetheless, "a government dismissed on the basis of grave accusations will be seen as discredited in the eyes of the population", he said.