The world number one ended the nine-time champion's reign 7-5 6-3 6-1.
The Serb, now two wins from completing the career Grand Slam of all four major titles, will play Andy Murray in the semi-finals on Friday.
Nadal, on his 29th birthday, suffered his first defeat in six years and his second in 72 matches at Roland Garros.
His only previous French Open loss came in the 2009 fourth round against Sweden's Robin Soderling - but this defeat was far less of a shock.
Djokovic, 28, went into the match as the favourite, on a 26-match winning streak and as a man on a mission, desperate to add the French Open to his Wimbledon, Australian and US Open victories.
"It's definitely a big win, a match that I will remember for a long time," he said afterwards.
"You go through more emotions than for any other match. Of course, playing against Rafa in Roland Garros, it's a special thing. It's a special match."
He added: "Tomorrow is a new day and I have to move on. It's only quarter-finals, and I want to fight for the title.
"That's what I came here for. I have to kind of direct my thoughts to the semis."
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