European ministers agreed on Monday to impose sanctions against 21 officials from Russia and Ukraine, including travel bans and asset freezes.
The measures come after Sunday's controversial referendum in Crimea, when 96% of voters backed leaving Ukraine and joining Russia, according to Crimean officials.
Crimea's parliament already declared independence from Ukraine on Monday and asked to join Russia.
Russian and Ukrainian politicians responsible for organising Sunday's referendum in Crimea are the main targets of the EU sanctions.
Kiev's government, as well as the EU and the US have condemned the referendum as illegal and have urged Russia to negotiate directly with Ukraine and to allow in observers and mediators.
"I don't have to remind any of you that it's illegal under the constitution of Ukraine and under international law. I call upon Russia yet again to meet with Ukrainian leaders and to start dialogue with them, and to try and move to de-escalation, as quickly as possible. We've seen no evidence of that." said EU Foreign Affairs Chief Catherine Ashton.
Further measures are expected to be taken later this week, when EU leaders meet in Brussels for the last scheduled EU Summit before the European elections.
The Crimean peninsula, home of 2 million people, has been under Russian military control since February.
