Phillip Cocu's side have built their impressive league campaign on an impeccable record at the Philips Stadion, but Ajax ended their 12-game winning run with a 3-1 victory.
Two goals in the closing minutes gave the visitors all three points, Lasse Schone and Anwar El-Ghazi on target to the delight of Frank de Boer.
Ajax's late strikes came after Luuk de Jong had cancelled out Ricardo Kishna's first-half opener, but PSV remain in control of the title race with a comfortable advantage.
Another turbulent week for Feyenoord ended in further disappointment as a goalless draw with Utrecht saw them fall further behind Ajax and PSV – piling the pressure on head coach Fred Rutten.
Midweek trouble in their UEFA Europa League clash with Roma brought the club back into the headlines for all the wrong reasons, and the players struggled under the spotlight as they failed to score away from home for a second successive league match.
At the bottom, Excelsior moved eight points clear of the danger zone after a comprehensive 3-0 victory over Heerenveen.
Luigi Bruins gave Excelsior an early lead after five minutes before Tom van Weert doubled their advantage early in the second half with his third goal in two games.
Kevin Vermeulen secured the victory in stoppage time, cutting in from the left to fire into the top corner.
Meanwhile, Xander Houtkoop rescued a point for ADO Den Haag with a stoppage-time strike that saw their match with Groningen finish 1-1.
Den Haag found themselves behind after 53 minutes when Tjaronn Chery converted from 12 yards, three minutes after team-mate Nick van der Velden had spurned a penalty attempt.
Groningen were unable to hold onto the win, though, as Houtkoop fired low past Sergio Padt five minutes into stoppage time.
