The document sets a legal framework for possible deployment of US troops in the country at a time of Russian aggression against Ukraine.
Czech Defence Minister Jana Cernochova signed the treaty together with US Defence Secretary Lloyd Austin in Washington, DC on May 23.
The US has similar agreements with 24 other NATO members, including Poland, Slovakia, Hungary, Lithuania, Latvia, Estonia, Romania and Bulgaria that form the eastern flank of the alliance.
Any deployment of US forces would still need approval by the Czech government and Parliament.