Czech President Vaclav Klaus has signed the Lisbon Treaty, becoming the last EU leader to sign the document into law.
The Czech Constitutional Court had earlier removed one of the last hurdles to its ratification by ruling that the Treaty is in line with the country's constitution.
Mr Klaus said: 'I announce that I signed the Lisbon Treaty at 15:00 (2pm Irish time) today.
'I had expected the court ruling and I respect it, although I fundamentally disagree with its content and justification.'
The Treaty aims to streamline decision-making in the 27-nation EU.
The ratification will mean that the union can pick its first-ever long-time president and a more powerful foreign representative.
The Czech Republic was the only EU country that had not yet ratified the accord, which needed the consent of all member states to come into force.
Mr Klaus earlier said he would raise no further objections after EU leaders agreed in Brussels last week to give the Czechs an opt-out from the Charter of Fundamental Rights.
President of the European Commission Jose Manuel Barroso said the treaty could be in force as early as December or January.
He said: 'The road has been a marathon of hurdles but last hurdle is now removed.'
Ireland voted in favour of the treaty at the second time of asking last month.
Taoiseach Brian Cowen said: 'This now clears the way for the treaty to enter into force in the near future.
'This is excellent news both for Ireland and for the European Union.'
In Britain, William Hague of the Conservative party said it was 'no longer possible' for an incoming Conservative government to hold a referendum on the treaty.