Mississippi's U.S. senators, Thad Cochran and Roger Wicker, along with Alabama's senators, Jeff Sessions and Richard Shelby, were among the Senate Republicans who blocked an effort by Democrats and the White House to lift the ban on gays from serving openly in the military.
The measure needed 60 votes to pass. Fifty-four Democrats voted in favor of the repeal. Forty Republicans and three Democrats opposed it.
The mostly partisan vote Tuesday was a setback for gay rights groups who saw the legislation as their best hope for repeal of the 17-year-old law known as "don't ask, don't tell."
Sessions says it's wrong to repeal "don't ask, don't tell" without completing a study of how it would affect troop readiness and morale.
Democrats included the repeal provision in a $726 billion defense policy bill, which authorizes a pay raise for the troops among other popular programs.