The EU's top two officials have sharply criticised Russia for putting pressure on Ukraine to drop plans to sign a trade and co-operation agreement with the EU this week.
The statement came amid new clashes in Ukraine's capital Kiev between pro-EU demonstrators and police.
"It is up to Ukraine to freely decide what kind of engagement they seek with the European Union," the EU said.
"We therefore strongly disapprove of the Russian position and actions."
The statement was issued jointly by European Council President Herman Van Rompuy and European Commission President Jose Manuel Barroso.
They insisted that stronger ties with the EU would not undermine Ukraine's existing relations with other neighbours, such as Russia.
"The Eastern Partnership is conceived as a win-win where we all stand to gain," they said.
Russia has threatened unspecified economic measures if Ukraine signs the deal with the EU. In the past Moscow has suspended gas supplies, in disputes over prices, and this year it blocked imports of Ukrainian chocolates.
Russia has also boycotted wine and mineral water from Georgia and Moldova - two other ex-Soviet states seeking favourable trade terms with the EU.
Georgia and Moldova are expected to initial agreements with the EU at an Eastern Partnership summit in Vilnius, Lithuania, on 28-29 November.
Ukrainian police have used tear gas in fresh clashes with pro-EU activists in the heart of Kiev.
Reports say some protesters tried to enter the government building but were pushed back by police.
Thousands of protesters gathered on European Square again after a huge rally on Sunday - the biggest since the 2004 Orange Revolution.
They are angry at the government's decision not to sign the trade and association deal with the EU.
Russia has urged Ukraine to join a Russian-led customs union with two other former Soviet states - Belarus and Kazakhstan.
Ukrainian opposition leaders, including world heavyweight boxing champion Vitali Klitschko, called the government's abrupt change of course "shameful" and vowed to keep up mass protests in central Kiev indefinitely.
The activists included supporters of jailed opposition leader Yulia Tymoshenko, who urged the government to release her - a plea that has also been made by EU politicians.
Ms Tymoshenko wants to get treatment in Germany for chronic back pain and the EU has made her release a key condition for signing the pact with Kiev. But last week Ukraine's parliament rejected legislation that would have enabled her to go.
More than 100,000 people rallied in Kiev on Sunday, the opposition said. Police estimated the crowd at about 50,000.
Hundreds of protesters stayed in tents on the square overnight, despite an order from the authorities not to do so.
Tents were a big feature of the pro-Western Orange Revolution, which challenged Russia's traditional influence in Ukrainian politics.