The proposed agreement sought to curb piracy, but internet campaigners said it posed a threat to online freedoms.
The rejection vote followed a failed attempt to postpone the decision because of ongoing investigations into Acta by the European Court of Justice.
Euro MP David Martin said: "It’s time to give [Acta] its last rites."
Twenty-two EU member states, including the UK, had signed the Acta treaty – but it had not been formally ratified.
Outside the EU, the treaty also had the support of the US, Australia, Canada, Japan, Morocco, New Zealand, Singapore and South Korea.
However, following significant protests, several countries chose not to back it.
Wednesday’s vote is seen by most observers as the final blow to the treaty in its current form. It means no member states will be able to join the agreement.
A total of 478 MEPs voted against the deal, with 39 in favour. There were 165 abstentions.
‘Hello democracy’
EU Trade Commissioner Karel De Gucht said work on tackling piracy would continue.
"Today’s rejection does not change the fact that the European Commission has committed itself to seeking answers to the questions raised by the European public," he said.
"The European Commission will continue to seek the legal opinion of the European Court of Justice on whether this agreement harms any of the fundamental rights of European citizens – including freedom of speech.