Striker Riga Mustapha (15th minute) gave Pune the lead with a penalty kick conversion before striker Miljan Mrdakovic (38th) pulled the hosts level in the first half. Second half strikes from medio Closa Gonzalez (61st) and Mrdakovic (69th) handed Tampines Rovers all three points.
With this loss, Pune FC slipped down to fourth place in Group-H behind leaders Kitchee SC (7 pts), Nay Pyi Taw FC (4 pts) and Tampines Rovers (3 pts) midway through the group-stage.
Pune coach Mike Snoei made four changes in the line-up handing starts to Mirjan Pavlovic, Anthony D'Souza, Mumtaz Akhtar and Gurjinder Kumar in place of Arata Izumi, Dhanpal Ganesh, Srikanth Ramu and Fanai Lalrempuia.
After soaking pressure in the opening exchanges, Pune FC took the lead at the quarter-hour mark.
As Pavlovic jumped to head a high rising ball inside the box, Tampines goalkeeper Hyrulnizam Juma'at fumbled and continued to head out hoping to control. In doing so, he ended up pulling down Pavlovic and the referee pointed to the spot.
At the other end, Tampines pulled level to restore parity. Keeper Amrinder Singh and skipper Anas Edathodika's mix up resulted in a back pass inside the penalty box and the referee awarded an indirect free-kick on the edge of the 6-yard box.
Skipper Mustafic Fahruddin's powerful free-kick was blocked by the wall but he did well to lob the ball to the far post for Mrdakovic who headed in from close range.
At the other end, Pune FC began to push up and went close through Pavlovic, Riga and Gabriel. However, poor finishing in the final third let the trio down as both sides went into the break tied at 1-1.
Pune FC brought in playmaker Lalrempuia Fanai at the start of the second half in place of medio Mumtaz Akhtar.
Controlling possession at the start of the second half, Pune FC almost took the lead at the hour mark. After some good work on the left, Gurjinder found Gabriel but his first timer from just inside box went over.
As Pune FC began to push up, the hosts took the lead at the other end. Defender Mohammad Shaiful found Gonzalez with a square pass from the left and the medio scored from 25-yards out to the right top corner of the net at the second post.
Eight minutes later, Tampines doubled the lead with a third goal. A move initiated by Aleksandar Duric from the left found Gonzalez on the right. Defender Srikanth Ramu's missed clearance allowed Mrdakovic to drive the ball home from close and complete his brace.
Minutes later, a great combination play in the midfield from defender Calum Angus and playmaker Douhou Pierre saw the Ivorian set up Pavlovic inside the box. The striker held off a defender before his shot on the turn was saved by the keeper.
Five minutes later, it was Riga who went close. However, the Dutchman's attempt from inside the box somehow sailed over the bar.
In the later stages of the game, Pune FC kept on pushing up, but were unable to capitalise on the chances they created as the game finished 3-1.