On a vote of 61-30, the Senate voted to move ahead on the legislation that would prohibit workplace discrimination based on sexual orientation and gender identity.
The bipartisan vote increases the chances that the Senate will pass the bill by week's end, but its prospects in the Republican-led House are dimmer.
Speaker John Boehner remains opposed to the bill, arguing that it will lead to frivolous lawsuits and undercut job creation.
A vote would come 17 years after the Senate rejected a similar discrimination measure by one vote.
The Obama administration has said passage of the bill is long overdue.