The treaty will bring into force the EU's plan to overhaul its institutions, elect its first-ever long-term president and choose a foreign policy high representative.
Fredrik Reinfeldt, the Swedish prime minister, whose country currently holds the rotating EU presidency, said the treaty could now come into force on December 1.
"Czech President Vaclav Klaus has signed the treaty. Thus the final hurdle in the way of the Treaty of Lisbon has been cleared," he said.
Describing himself as "very pleased" at Klaus' move, Reinfeldt said the signing ended "far too long period of institutional focus within the EU".
"It opens up [the way] for a more democratic, transparent and efficent union," the prime minister said.
Reinfeldt said he would call an EU summit shortly and would "now begin name consultations" among the 27 member states on who to pick for the presidential and foreign policy roles.
Javier Solana, the EU foreign policy chief whose job will disappear under the treaty, said its entry into force "will enable us to work in a much more coherent and efficient way and give us a stronger voice on the international scene".
Gordon Brown, the British prime minister, welcomed Klaus' signature as "an important and historic step for all of Europe".
Britain's opposition Conservative Party said it would now drop plans to call a referendum on the treaty if it takes power following next year's general election.
William Hague, the party's foreign affairs spokesman, said: "Now that the treaty is going to become European law and is going to enter into force, that means a referendum can no longer prevent the creation of the president of the European Council, the loss of British national vetoes.
"These things will already have happened and a referendum cannot unwind them or prevent them."
Klaus had raised concern over the pact, saying it could turn the EU into a super state with little democratic control.
The president sought an opt-out from the treaty nearly three weeks ago, after Prague's parliament had earlier ratified it, in an attempt to ensure the treaty would not allow ethnic Germans expelled from former Czechoslovakia after the second world war to reclaim their property.
Hamish Macdonald, Al Jazeera's correspondent in London, said the treaty will "pave the way for some pretty enormous changes".
"Perhaps the most high-profile of which will mean there will now be an EU president, a figurehead that is effectively the chairman of the EU council," he said.
"It will also give the European Union a senior foreign policy position, if you like a foreign minister, that will deal with things like Iran and the nuclear debate, Russia and also Afghanistan."
The EU president, who needs to be elected unanimously, will serve a two-and-a-half-year term, strengthening the current system of a six-month presidency that states hold in turn.