It was certainly a big shock for promotion-chasing Tranmere, who looked to be well on course as they took a two-goal lead inside five minutes.
But then errors crept into their play and they were made to pay the penalty.
Chris Greenacre fired Tranmere ahead inside two minutes following a cross by the lively Chris Shuker which was not cleared by Ian Cox.
Then in the fifth minute, Shuker deceived Gillingham keeper Dean Brill with a corner which crept inside the far post.
Tranmere were rampant at this stage but a couple of chances went begging before the visitors pulled a goal back out of the blue in the 11th minute.
Gary Mulligan was first to react after a header by Bas Savage rebounded off the bar.
Mulligan found the target again in the 19th minute following a long throw-in by Savage, taking full advantage of a mix-up involving keeper Gavin Ward and Ian Goodison.
Remarkably Gillingham took the lead in the 42nd minute. Ben Chorley netted his first goal of the season with a well-placed header following a deep cross by the hard-working Savage.
Kevin Ellison was just wide for Tranmere ahead of the break and a lot of second-half pressure brought no reward for the home side.
A Gareth Taylor header was saved at the second attempt by Brill. Shuker fired wide and then was off target with an angled free-kick after 54 minutes.
Brill saved confidently from Greenacre in the 70th minute and two minutes later substitute Steve Davies missed a big opportunity for Tranmere. He fired across the face of the goal when he should have done better after good work by Goodison.
Gillingham were not without their chances on the break but Matthew Jarvis saw an angled shot saved, Ward denied Mulligan and then skipper Mick Flynn missed the target with a 30-yarder.
Rovers sent on Calvin Zola as an extra striker late on but Gillingham continued to defend superbly with excellent work from Cox and Chorley.