Prime Minister Zurab Noghaideli said the move effectively put a stop to exports of Georgian goods to Russia.
Russia says construction work said the border was being closed for construction work.
The governor of the Georgian border region, Vassily Maglaperidze, said the closure of the Verkhny Lars checkpoint was politically motivated.
Maglaperidze also complained that the Russians had not warned that checkpoint was to be closed, as is customary practice. As a result, hundreds of cars are stuck at the border.
In a statement, the Georgian Foreign Ministry decried the move as "an unfriendly and provocative step."
Relations between Moscow and Tbilisi have grown increasingly tense lately.
In recent months Russia has banned Georgian wine and mineral water and indicated that it may recognize the claims to sovereignty made by two breakaway Georgian regions.