The move came as the Senate worked to wrap up loose ends on the authorization bill (S. 1050), which it passed last month.
Sens. Trent Lott, R-Miss., and Byron Dorgan, D-N.D., offered the amendment, calling the Pentagon's plan to mothball as many as one-quarter of its facilities outdated.
"Many of the threats we face today were not anticipated in 2001, when this round was approved," Dorgan said at a news conference.
The two senators said reducing the number of bases and consolidating forces might make it easier for terrorists to strike.
The amendment failed on a 53-42 vote.
Democrats in the House last month failed in a bid to block the next round of closings during debate on the House version of the defense authorization bill, although language was added that would require the Pentagon to keep open enough bases to allow for a future expansion of U.S. forces.
Supporters of that language said the Pentagon would have to close fewer bases in 2005 to comply with the troop strength provision.