Ukraine, Georgia and Moldova have signed partnership agreements with the European Union, in a move strongly opposed by Russia.
The pact – which would bind the three countries more closely to the West both economically and politically – is at the heart of the crisis in Ukraine.
Russia said that while the signing of the deal was the right of any state there could be grave consequences.
A ceasefire with pro-Russian rebels in east Ukraine is due to end on Friday.
Ukrainian President Petro Poroshenko hailed the signing as Ukraine’s most historic day since independence in 1991, describing it as a “symbol of faith and unbreakable will”.
“Ukraine is underlining its sovereign choice in favour of membership of the EU,” he said.
Meanwhile European Council President Herman van Rompuy described it as a “great day for Europe”.
But Russian Deputy Foreign Minister Grigory Karasin told Interfax news agency that the move was fraught with difficulties.
“The signing of this serious document is, certainly, a sovereign right of each state,” he said.
“[But] the consequences of the signing by Ukraine and Moldova no doubt, will be serious.”
Earlier senior Kremlin adviser Sergei Glazyev described Mr Poroshenko as a “Nazi” and said his presidency was illegitimate because parts of Ukraine did not vote in the May elections.
He also said that Mr Poroshenko had no constitutional right to sign the treaty, which would damage the Ukrainian economy.
However, President Vladimir Putin’s spokesman Dmitry Peskov told Russian media that Mr Glazyev’s comments did not reflect the official Kremlin position.
Mr Poroshenko’s predecessor Viktor Yanukovych refused to sign the deal under pressure from Russia and protests led to his overthrow.
After this Russia annexed Ukraine’s Crimea region and pro-Russia separatists in eastern regions declared independence, claiming that extremists had taken power in Kiev.
Fighting is said to have continued in some areas of eastern Ukraine despite a temporary ceasefire this week.
Talks on extending the truce in in the Donetsk and Luhansk regions are also set to take place on Friday.
In another development, rebels released four international observers captured more than a month ago.
Alexander Borodai, head of the self-styled Donetsk People’s Republic, said the members of the Vienna-based Organisation for the Security and Co-operation in Europe (OSCE) had been freed as a goodwill gesture.
More than 420 people have been killed in fighting between pro-Russia rebels and government forces in eastern Ukraine since mid-April, the UN estimates.