The Wirral side started the game hovering just above the relegation zone, but they will make the long trek home in celebratory mood after a hard-earned success at fellow strugglers Colchester.
Ryan Lowe pounced to grab Rovers' precious winner, just after the hour-mark, after Freddie Sears had cancelled out Jean-Louis Akpa Akpro's opener.
Tranmere took the lead nine minutes before the break with a goal against the run of play.
Colchester were caught in possession and Max Power fed dangerous wide man Akpa Akpro, who burst forward before slotting past goalkeeper Sam Walker.
It was hard luck on the U's, who had dominated proceedings prior to that setback.
Jabo Ibehre fired over and then burst through on goal after a clever pass from Sears - only to have his weak effort saved by Tranmere goalkeeper Owain Fon Williams.
Alex Gilbey then had a half-volley tipped over by the impressive Williams, who then came to his side's rescue again to keep out a rising effort from Sears.
But there was nothing the Rovers goalkeeper to do to keep out Sears' leveller.
Dominic Vose fed Ryan Dickson, who squared from the left, and Sears nipped in to turn the ball home from close range.
But i was the visitors who scored a precious winner.
Akpa Akpro beat right-back Gavin Massey, before delivering a low cross that was turned in by Lowe.