Trent Hunter, Jason Blake and Viktor Kozlov scored for the Islanders after spotting the Penguins a 1-0 lead. The Islanders moved one point ahead of Toronto and into 10th place in the Eastern Conference playoff race. They trail Carolina and Montreal - at the playoff cutoff - by one point but have games in hand.
New York returned home following an 0-3-1 road trip.
Colby Armstrong scored and Marc-Andre Fleury stopped 32 shots for the Penguins, who have lost consecutive games in regulation for the first time since Jan. 9-10.
The Islanders also halted Pittsburgh's 16-game point streak with a 6-5 victory on Feb. 19. The Penguins remained two points behind New Jersey in the Atlantic Division race.
Penguins 19-year-old phenom Sidney Crosby was held scoreless for the second straight game following a six-game point streak.
Both goalies made big saves in the first period to keep the game scoreless.
DiPietro got his stick down on the goal line to stop Michel Ouellet's rebound try at 9:56, and Fleury kicked out his left pad to stop Miroslav Satan during an Islanders power play at 13:45.
Armstrong finished a 2-on-1 with Gary Roberts by redirecting Roberts' cross-ice pass past DiPietro to give the Penguins a 1-0 lead at 16:55.
New York tied it at 2:37 of the second on Hunter's power-play goal. Satan quickly fed the puck to Hunter in the slot for a wrist shot that beat Fleury and gave the Islanders forward 20 goals this season.
The Islanders took a 2-1 lead at 5:43 when Blake sent an uncontested wrist shot from inside the left circle past Fleury.
DiPietro held the lead when he made a right pad save on Evgeni Malkin's first NHL penalty shot at 6:26. The puck trickled toward the goal line, but DiPietro smothered it with his glove before it went in. Malkin was given the chance when he was hooked down by defenceman Tom Poti on a breakaway.
Kozlov scored his 22nd goal at 1:42 of the third period, tying his career high.
Notes: Islanders forward Jason Blake played in his 499th NHL game. ... Pittsburgh is 8-3-1 in March. The Penguins' first two losses in the month were by one goal.