A powerful committee of the US House of Representatives has voted to block a deal that would give a Dubai-based company control over key US ports.
Mr Bush has given unqualified support to the deal
The vote is a direct challenge to President George W Bush by members of his own Republican party in Congress.
Lawmakers say they are concerned about the security implications of the deal - which is unpopular with the public.
Dubai Ports World is taking over P&O, which runs six major US ports from New Jersey to New Orleans.
The House Appropriations Committee voted 62-2 for an amendment to block the deal.
The full House is expected to confirm the committee's decision next week and the measure is also expected to be approved in the Senate.
The deal is currently in limbo while the US conducts a review.
Mr Bush has strongly backed the $6.85bn (£3.94bn) deal, saying repeatedly that he would veto any law designed to block it.
But opponents have made the legislation part of an emergency spending bill that provides extra funds for hurricane disaster relief and the wars in Iraq and Afghanistan - which the president would be hard pressed to reject.
House of Representatives to vote on amendment next week
Democrats pressing for Senate vote on issue
Measure goes to president for signature if both houses pass it
President says he will veto it
Congress can overrule veto with a two-thirds majority
"We want to make sure that the security of our ports is in America's hands," Rep Jerry Lewis, a California Republican, said before the vote.
Opinion polls have shown an overwhelming majority of Americans think the White House is wrong to back the deal.
With an election due in November and the popularity of both the president and Congress low, some Republicans apparently fear that Democrats could use the deal against them.
"We're not going to let the Democrats get to the right of us on national security," Rep Peter King, who chairs the homeland security committee, said recently.
The head of DP World said the deal would not give the company control of security at the ports.
Security "is in the hands of the American government. This includes the border security guards, customs officers and the US ports Authority. Our role is only loading and offloading goods," Sultan Bin Salim told the BBC Arabic service.
DP World is a state-owned company based in the United Arab Emirates, a US ally.
It is moving to take control of UK-based P&O, which holds contracts to run five ports in the US: New York, New Jersey, Philadelphia, Baltimore, Miami and New Orleans.
It also runs a container port at Vancouver in Canada.
President Bush has never vetoed a law in more than five years in office.
Congressional leaders reportedly believe they would have enough votes to override a presidential veto.
The president has not commented on the committee vote, but did criticise Congress on another matter on Wednesday.
Visiting New Orleans, he called for more money for the city - saying it had been "short-changed" by Congress after Hurricane Katrina.
"Congress heard our message about improving the levees, but they short-changed the process by about $1.5bn (£860m)," he said, referring to money it had cut for rebuilding the city's flood defences.
He also said a package of redevelopment aid should be focused on the city and not divided among all the Gulf Coast states affected by the storm.
Congress is expected to reconsider New Orleans' aid needs soon.