Peers have rejected a decision to build the UK's first super-casino in Manchester by just three votes.
The panel recommended a super-casino be built in east Manchester
It means the plans will not be implemented, even though MPs backed the proposal by a majority of 24.
The Lords vote also means that plans for 16 smaller casinos around the UK will have to be shelved.
Culture Secretary Tessa Jowell said that, after the defeat, she wanted "to reflect on the outcome" and promised new proposals.
She told BBC Radio 4's World Tonight: " When you're going to win a vote, you don't have a plan B, you go out to win a vote."
Ms Jowell added that she would now consider "the various options that are open to us".
Peers rejected the Gambling Order backing Manchester by 123 votes to 120, while MPs supported it by 274 to 250.
It is thought likely the government will say the verdict of the Commons - the elected house of Parliament - should rate above that of the Lords.
This could lead to a stand-off, with ministers sticking to the original proposals.
Richard Caborn, the minister for gambling, denied the defeat was humiliating.
"We will reflect on what the Lords have said and if we can address some of their issues we will come back and report that to Parliament," he said.
Shadow culture secretary Hugo Swire said: "It would be unthinkable for the government to attempt to bring back these same proposals."
Liberal Democrat Lord Clement-Jones said: "Against all the odds, this is an historic victory."
He added: "Ultimately, this is a triumph for Parliament and will ensure public confidence in its scrutiny function and ability to hold the government to account."
Manchester was picked earlier this year by an independent panel ahead of the front-runners Blackpool and the former Millennium Dome in London.
Some critics argued that Blackpool was a more suitable venue, with greater need for a super-casino.
Others said it could increase problem gambling in a deprived part of Manchester.
During the Commons debate, Blackpool North and Fleetwood Labour MP Joan Humble said her town had been unfairly "marked down" by the Casino Advisory Panel.
But supporters of the Manchester bid said the chosen area in the east of the city was in need of the investment and renewal the project would bring.
Manchester Central Labour MP Tony Lloyd said opposing the plans would be a "vote against Manchester".
The Lords vote means the super-casino plans will have to be re-drafted and that those for eight more "large" casinos and a further eight "small" ones are also thrown out.
Liberal Democrat culture spokesman Don Foster told the BBC that the votes for the super-casino and the other venues should have been separate.
He added: "It comes down to the real need for more scrutiny of the super-casino."
Earlier, Ms Jowell tried to head off a rebellion by accepting an amendment tabled by Labour peer Baroness Golding, setting up a new joint committee of MPs and peers to look at the lessons to be learnt from the casino selection process.
Large casinos had been due to be built in Great Yarmouth, Hull, Leeds, Middlesbrough, Milton Keynes, Newham, Solihull and Southampton.
The sites chosen for smaller venues were: Bath and North East Somerset, Dumfries and Galloway, East Lindsey, Luton, Scarborough, Swansea, Torbay and Wolverhampton.