Two goals from former Tranmere loanee Jennison Myrie-Williams knocked Rovers out of the Johnstone's Paint Trophy on Tuesday night.
The winger fired in a stunning free kick against his former club early in the second half and added the second from the penalty spot with 15 minutes remaining to complete a 2-0 win for Port Vale.
There were four changes to the team that defeated Colchester 4-0 with Joe Thompson returning to the side and Adam McGurk, Danny Harrison and Max Power making their first starts of the season as James Wallace, Andy Robinson, Liam Palmer and Jean-Louis Akpa Akpro all rested. Former Rovers midfielder Chris Shuker started in the centre of midfield for Port Vale.
There were chances at both ends in the first ten minutes. Vale attacked straight from the kick off and tested Owain Fon Williams before Max Power sent a volley straight at Chris Neal.
Cole Stockton cut his way through the Vale defence before dragging a right foot shot wide of goal while Myrie-Wiliams headed over from a corner and Louis Dodds fired past the left hand post.
After a Myrie-Williams shot had been ruled out for offside, it needed a strong header from Clayton McDonald to prevent Thompson from getting a free header at the back post.
Zoumana Bakayogo put the ball in the Port Vale net from eight yards on 26 minutes from an Abdulai Bell-Baggie cut back but the flag had already been raised for offside.
Adam McGurk shot wide of the target from 20 yards on 27 minutes while Dodds missed to the left of Fon Williams' goal from a similar distance at the other end.
Fon Williams saved twice from Dodds at his near post around the 34 minute mark with the striker heading on to the roof of the net with a follow up effort on the second occasion.
McGurk latched on to a long ball from half time substitute Michael Kay on 53 minutes but his first touch took the ball slightly too far and Neal was able to gather on the edge of his area.
A foul by Bakayogo 25 yards from goal gave Vale a shooting opportunity on 57 minutes and the home team took full advantage. Both Shuker and Myrie-Williams stood over the ball and it was the latter who took the set piece, firing an unstoppable shot past Fon Williams and into the top left corner.
Cole Stockton was introduced in place of Thompson on 65 minutes, a change that saw McGurk move back out to the left and soon after his shot was deflected away from goal for a corner.
However, the action was soon back in the Rovers half and Vale doubled their lead from the penalty spot. A cross was whipped into the box with Roman Golobart crouching to control only for the ball to strike his arm. Myrie-Williams send Fon Williams the wrong way for his second of the evening.
After Harrison's volley from a corner missed the target on 80 minutes, a Bell-Baggie run came to an end inside the area when the winger was marshalled away from goal.
Myrie-Williams' attempt to complete his hat-trick from outside the area went narrowly wide of the target on 81 minutes while a late McGurk effort curled away from goal.
Port Vale: Neal, McCombe, Loft, Dodds (Williamson 70), Myrie-Williams, Vincent, McDonald, Shuker, Morsy (Burge 56), Duffy (Yates 70). Substitutes: Taylor, Lloyd.
Tranmere: Fon Williams, Holmes (Kay 45 HT), Golobart, Gibson, Bakayogo, Bell-Baggie, Power (Kirby 75), Harrison, McGurk, Thompson (Stockton 65), Cassidy. Substitutes: Mooney, McChrystal.
Referee: Stephen Martin
Yellow cards: Morsy (29)