Though the revolt was defeated, it could undermine Cameron's leadership, as scores of his own party's lawmakers took the highly unusual step of voting to criticise his government's legislative plans, a week after they were first put before Parliament.
The rebels are angry that the government's policy proposals did not include steps to make Cameron's promise of a referendum on Britain's EU membership legally binding.
The party turmoil has fuelled talk of Britain sliding towards the EU exit and has stirred memories of Conservative infighting that contributed to the downfall of former prime ministers Margaret Thatcher and John Major.
While the vote was non-binding, the scale of the mutiny, less than two years before the next parliamentary election, will embolden eurosceptics pushing him to take a harder line on Europe.
A total of 130 lawmakers supported an amendment expressing regret that the EU referendum was left out of the government's agenda. Of those, 114 of the Conservatives' 305 members of parliament voted against Cameron.
Senior Conservatives put on a brave face after the largely symbolic ballot.
"When all the dust has settled, there is one essential fact: one party, the Conservative Party, is committed to a referendum on leaving or staying in the European Union, and the other parties are not," Foreign Secretary William Hague said.
Just before the parliamentary ballot, Cameron played down its significance, saying he was "extremely relaxed".
Cameron had hoped to end party squabbling over Europe in January when he promised to renegotiate Britain's relationship with the EU and hold a referendum on its membership before the end of 2017, provided he wins the next general election in 2015.
But Conservative eurosceptics soon began pushing for a law before 2015 to guarantee the referendum would take place. Some even called for an earlier referendum.
Cameron's offer on Tuesday of draft legislation that would make his pledge legally binding received a lukewarm reception. Rebels say it will be blocked by the Conservatives' coalition partner, the pro-EU Liberal Democrats.
Wednesday's parliamentary vote underscored how Cameron is boxed in over Europe.
Keen to avoid a rift with the Liberal Democrats, he must also avoid alienating Conservative eurosceptics who see the EU as an over-mighty "superstate" that threatens Britain's sovereignty.
The success of the anti-EU UK Independence Party in local elections this month only intensified Conservative pressure for Cameron to go further on Europe. A YouGov poll in April put support for withdrawal at 43%, with 35% wanting to stay in.