The EU signed the deal hours after announcing more targeted sanctions.
Pro-Moscow leader Viktor Yanukovych's abandonment of the deal in November had led to deadly protests, his removal and Russia taking over Crimea.
On Friday, Russia's upper house unanimously approved the treaty on Crimea joining the Russian Federation.
'Rule of law'
The EU Association Agreement is designed to give Ukraine's interim leadership under PM Arseniy Yatsenyuk economic and political support.
EU President Herman Van Rompuy said in a statement that the accord "recognises the aspirations of the people of Ukraine to live in a country governed by values, by democracy and the rule of law".
Mr Yatsenyuk said: "I strongly believe the EU will speak with one single strong voice, protecting Ukraine."
He added that "the best way to contain Russia is to impose real economic leverage".
The BBC's Matthew Price in Brussels says Friday's accord is not the full package that Mr Yanukovych rejected in November - many parts will not be signed until after new presidential elections in May.
The most sensitive issue of trade integration with the EU is as yet unsigned.
But in its Conclusions on Ukraine, published on Friday, the EU said it was committed to signing the remainder.
The EU also said it would push ahead with similar cooperation deals with two former Soviet republics - Georgia and Moldova - in the summer.
Moscow has troops in breakaway parts of Georgia and in Trans-Dniester, which broke away from Moldova, and our correspondent says the EU's announcement on this is likely to infuriate the Kremlin far more than any sanctions imposed so far.
The EU has also cancelled a summit with Russia in June and said member states would cancel regular bilateral summits.
Friday's signing came hours after the EU broadened its sanctions over Russia's annexation of Crimea.
It added 12 individuals to an earlier list of 21 who now face asset freezes and travel bans.
The US on Thursday added to its own list and also targeted the Rossiya bank.
Russian Foreign Minister Sergei Lavrov said on Friday the international sanctions were "absolutely unlawful".
However, President Vladimir Putin said after talks with officials in Moscow that Russia would not take an immediate reciprocal action.
"I think we should refrain from taking steps in response for now," Interfax quoted Mr Putin as saying.
However, Russian Prime Minister Dmitry Medvedev said Ukraine should pay back $11bn in gas discounts as an agreement linked to the lease of the Sevastopol naval base in Crimea was now invalid.
In response to the US sanctions on Rossiya bank, Mr Putin said: "I personally did not have an account with it but will open an account there on Monday for sure."
Two credit rating agencies have now downgraded Russia's outlook to negative from stable.
In Moscow, all 155 senators present in the upper house of parliament voted to ratify the treaty incorporating Crimea into the Russian Federation.
Mr Putin is expected to complete the process by signing the treaty at a ceremony later on Friday.
- 21 Nov 2013: President Viktor Yanukovych abandons an EU deal
- Dec: Pro-EU protesters occupy Kiev city hall and Independence Square
- 20-21 Feb 2014: At least 88 people killed in Kiev clashes
- 22 Feb: Mr Yanukovych flees; parliament removes him and calls election
- 27-28 Feb: Pro-Russian gunmen seize key buildings in Crimea. Parliament, under siege, appoints pro-Moscow Sergei Aksyonov a PM
- 6 Mar: Crimea's parliament votes to join Russia
- 16 Mar: Crimea voters choose to secede in disputed referendum
- 17 Mar: Crimean parliament declares independence and formally applies to join Russia
- 18 Mar: Russian and Crimean leaders sign deal in Moscow to join the region to Russia
Source: BBC News