The Eurosceptic Czech president opposes the treaty
The lower house of the Czech parliament has approved the EU's Lisbon Treaty - a key step towards ratification.
The treaty has not yet been approved by the upper house, the Senate, where its passage is likely to be further delayed by right-wing opponents.
The Czech Republic - current holder of the EU's rotating presidency - is among a handful of countries that have not yet ratified the reform treaty.
It was rejected by Irish voters in a referendum last June.
It cannot take effect unless all 27 EU member states ratify it.
The Republic of Ireland government plans to hold a new referendum on the treaty this year, having secured sovereignty "guarantees" from EU leaders.
On Wednesday the Czech lower house voted 125 to 61 to adopt the document, aimed at streamlining EU institutions to make them more flexible after the 27-nation bloc's enlargement in recent years.
Czech President Vaclav Klaus has argued that the treaty would undermine Czech sovereignty.
LISBON TREATY PROGRESS
Approved by parliament: Austria, Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Lithuania, Luxembourg, Malta, The Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, UK
Defeated by referendum: Irish Republic
Challenges: Legal objections delaying ratification in Germany, Polish president also delaying ratification, Czech upper house yet to vote on treaty
The treaty is seen by opponents as a way to impose a federalist agenda, undermining national sovereignty.
Czech Prime Minister Mirek Topolanek, leader of the conservative Civic Democratic Party (ODS), has signed the treaty, but a substantial part of his own party opposes it.
There is pressure from some Czech politicians to delay ratification until parliament backs the plan to host a radar base for a US missile defence shield.
The Republic of Ireland was the only EU member state to hold a referendum on Lisbon. Other governments argue that it is an amending treaty which does not change the EU enough to justify a referendum.
In Germany the treaty has gone before the constitutional court. Poland's President Lech Kaczynski is also delaying ratification, insisting that the Irish battle over Lisbon must be resolved first.