Barnsley maintained their promotion push with a hard-earned victory.
The winner came in the 15th minute when Chris Shuker crossed for the unmarked Paul Heckingbottom to head powerfully into the top right-hand corner.
Delroy Facey, Mike Jackson and Sam Aiston all wasted good chances for Tranmere as the home side hit back.
Substitute Steve Davies fired in a powerful effort late on but Nick Colgan was equal to the task as the Tykes inflicted Tranmere's first loss in 12.
Tranmere: Seremet, Francis, Jackson, Goodison, Roberts, Raven, Rankine (Jennings 66), O'Leary, Aiston (Davies 66), Facey, Greenacre.
Subs Not Used: Wilson, Harrison, Tremarco.
Booked: Jackson, Raven.
Barnsley: Colgan, Hassell, Kay, Austin, Heckingbottom, Shuker, Burns, McPhail, Devaney (Williams 89), Richards (Nardiello 77), Wright (Hayes 61).
Subs Not Used: Flinders, Howard.
Booked: Wright, Burns, Nardiello.
Goals: Heckingbottom 16.
Att: 6,802.
Ref: N Swarbrick (Lancashire).