The document was inked by Lithuanian Defence Minister Raimundas Karoblis and Anne Hall, the US ambassador to Lithuania.
The minister said that the signing of the agreement marked "a historic day for Lithuania-US defence cooperation".
"I am confident that US troops' presence here is a key factor that allows people in Lithuania to feel safer in today's security situation and I hope that the constant presence will continue in the future," he said during the ceremony.
The US ambassador said that the agreement "reaffirms the two countries' mutual commitment to work side-by-side strengthening the NATO alliance".
The US was also to sign such an agreement with Estonia on Tuesday after signing one with Latvia last week.
Baltic and US negotiators aimed to finalize the agreements before the inauguration of US President-elect Donald Trump as the arrival of the new administration might push the issue to the bottom of the political agenda.
The bilateral agreements on defense cooperation, known among military officers and diplomats as "bilateral SOFAs", are drafted at the US initiative. They define the status of US troops and their families. The agreement with Lithuania will take effect after it is ratified by the country's parliament.
The agreement gives the US jurisdiction over crimes committed by its military personnel, but allows Lithuania as the host nation to take over jurisdiction in cases of particular importance to it. The document also gives the US the right to use certain military facilities, although these facilities remain in the ownership of Lithuania.
A company-sized US unit has been stationed in Lithuania since in 2014 and a battalion from the US brigade that is being deployed to Poland is expected to frequently come to Lithuania for training.