The move, formally confirmed Thursday, will allow French prosecutors to investigate Le Pen over tweets she sent in December 2015 that showed images of killings by ISIS militants.
French law prohibits the distribution of violent images or incitement of terrorism.
Announcing the result of the vote on whether to lift the immunity, EU Parliament President Antonio Tajani said a "clear majority" of members backed the motion.
Le Pen told CNN she's not worried about her immunity being lifted.
"I notice that freedom of expression of an EU member of Parliament who denounces the actions of ISIS allows the French government to take her to court," she said. "I will express myself in court and say what I think of all this. ... "
As a member of the EU Parliament, Le Pen, leader of the National Front, enjoys immunity that covers freedom of speech -- but that immunity can be lifted if authorities of the person's member state makes a request.
In this case, a prosecutor in Nanterre, in the west of Paris, made the request.
Under French law, the maximum penalty for distributing violent images is three years in prison and a fine of up to 75,000 euros (about $79,000).
The lifting of Le Pen's immunity relates to this case only, and any action is unlikely to occur before the first round of voting in the French presidential election on April 23.