The House Appropriations Committee voted by 62-2 on Wednesday to bar DP World, run by the government of Dubai in the United Arab Emirates, from holding leases or contracts at US ports.
The vote underscored widespread public opposition to the deal and Republican fears of losing its edge on the issue of national security in November's elections.
The panel's vote came as across the Capitol, Democrats clamoured for a vote on the same issue in the Senate.
George Bush, the US president, has promised to veto any legislation that would stymie the deal.
By its vote, the House committee attached the ports issue to a $91 billion measure to finance the recovery in southern states from Hurricane Katrina and the wars in Iraq and Afghanistan - a measure that must be passed.
The full House could consider that measure as early as next week.
For now, Republican Senate leaders hope to delay an early showdown with Bush on the issue.
"Whoever's responsible for those ports of entry should be American"
Bill Young, chairman of the House defence appropriations subcommittee
But by adding the provision to the measure paying for the war and hurricane relief, the House panel illustrated a willingness, even by members of Bush's own party, to defy him on the issue.
Bill Young, chairman of the House defence appropriations subcommittee, said: "One of the most vulnerable situations facing America is our ports of entry.
"Whoever's responsible for those ports of entry should be American."
Representative Marcy Kaptur said allowing the DP World takeover to continue and ignoring public outcry over it would be irresponsible.
"The American people elected us to do something when an issue like this comes up," she said.
Only Jim Kolbe, a Republican, and Jim Moran, a Democrat, voted against the measure.
Moran said: "It is premature, we don't have enough information and ... it may turn out to be unnecessary."
The developments showed the extent to which the politically charged issue has come to dominate the agenda in recent days, with Republicans and Democrats competing to demonstrate the strongest anti-terror credentials in the run-up to mid-term elections.