Lib Dem frontbencher Edward Davey has been ordered out of the Commons, after angry protests to the deputy speaker.
Mr Davey was annoyed at the decision not to allow MPs to debate and vote on a Lib Dem call for a referendum to be held on the UK's membership of the EU.
Deputy Speaker Sir Michael Lord acted after Mr Davey defied warnings. Fellow Lib Dems then walked out in support.
The Lib Dems support the EU treaty and UK membership of the EU. They oppose a referendum on the treaty itself.
The protest came as MPs began the latest day of debate on the Lisbon Treaty - something the Conservatives, some Labour and some Lib Dem MPs have said should be subject to a referendum.
Points of order
The Lib Dem leadership, who say they would not vote for such a referendum, say the whole issue of Britain's membership of the European Union should be subject of a referendum instead.
They had put down an amendment to the EU treaty debate to that effect.
But when deputy speaker Michael Lord told MPs that Speaker Michael Martin had ruled the amendment would not be debated - Lib Dem MPs raised a series of points of order in protest.
Mr Davey, the party's foreign affairs spokesman, complained he was being "gagged" and added: "We need to know when we will debate this issue. It's a debate the British people want."
Referring to the ruling he said: "It is an outrage to this House." But Sir Michael told him: "The outrage to the house is in danger of being the honourable member's attitude to the chair."
When he refused to sit down to allow the debate to start, Sir Michael lost his patience and ordered him out.
The Kingston and Surbiton MP will no longer be able to take part in any of the remaining proceedings or votes on Tuesday.
Sir Michael also warned other Lib Dem MPs raising points of order - including leader Nick Clegg and president Simon Hughes - that they should respect the decisions of the occupier of the Speaker's chair.
Deputy speaker Sir Michael Lord warned Mr Davey several times
The challenge to the Speaker's decision comes at a time when Speaker Michael Martin has found his position under pressure.
At the last general election in 2005 all three main parties promised a referendum on the planned European Constitution.
The government argues that the new EU treaty is significantly different from the constitution - which was rejected by French and Dutch voters - and does not require a referendum.
Conservative MP John Redwood told MPs it was a "discourtesy" that the Lib Dems walked out when important issues were up for debate "after synthetic anger about their broken promises".
And Neil O'Brien, director of the I Want a Referendum campaign - which is pushing for a referendum on the treaty, said: "The Liberal Democrats are spitting mad because they have been rumbled."
"They wanted to camouflage the fact that they have chickened out of a referendum on the constitutional treaty by calling for a referendum on Britain's membership of the EU.
"But now it has become apparent that this "in or out" referendum is not even going to be discussed."