Tranmere's ninety four years in the Football League was ended by a 3-2 defeat at Plymouth Argyle.
Rovers' loss combined with Hartlepool's win relegated Rovers from League 2.
Alan Rogers made five changes to the team that started against Oxford. Into the side came Jason Koumas, Marc Laird, Adam Dugdale, Matt Hill and Rory Donnelly, with Iain Hume, George Green, Jennison Myrie-Williams and injured pair Danny Holmes and Rob Taylor dropping out.
Backed by a fantastic noisy following behind the goal, Rovers started well and saw Donnelly force a save out of Plymouth keeper Luke McCormick from Kayode Odejayi's flick on.
However, on six minutes the home side won a penalty when Max Power was ruled to have fouled Dominic Blizzard, with Reuben Reid sending Owain Fon Williams the wrong way to put the Pilgrims 1-0 up.
Donnelly was inches away from connecting with a right wing cross from Jason Koumas on 11 minutes while Curtis Nelson had a header saved by Fon Williams at the other end four minutes later.
Koumas tested McCormick with a low 25 yard strike on 17 minutes while the keeper did well to save from Donnelly soon after when the Swansea City loanee had been sent through by Laird.
Plymouth launched a quick counter attack on 21 minutes and saw Lewis Alessandra fire over the Tranmere goal from a tight angle and Fon Williams had to be alert to cut out a through ball for Reid six minutes later.
Rovers started to dominate possession and a long spell inside the Plymouth half paid off on 34 minutes. Michael Ihiekwe's cross was turned behind for a corner and after Odejayi stepped over a low Koumas corner, Power curled into the top left corner from 15 yards.
However, two minutes later Rovers were behind once more. A corner was cleared outside the Rovers area but Donnelly lost possession to Tareiq Holmes-Dennis who fired into the bottom left corner from 25 yards.
Fon Williams parried Reuben Reid's left foot shot three minutes into the second half and a long range effort from Curtis Nelson deflected a couple of yards past the left hand post soon after.
Donnelly fired over the top right corner from 20 yards on 52 minutes while a Fon Williams clearance was charged down by Alessandra at the other end but the ball deflected wide of goal.
George Green was introduced in place of Laird on 59 minutes and Iain Hume came on for Hill soon after as Rogers changed Rovers' attacking threat.
Fon Williams pulled off a superb one handed save to deny Alessandra on 67 minutes, the keeper reacting quickly to palm a close range header behind for a corner.
Rovers were agonisingly close to an equaliser on 75 minutes, with a Koumas cross from a clever free kick routine deflecting over McCormick only for Carl McHugh to head off the line at the back post.
Odejayi held off a defender and laid the ball off to Hume on 77 minutes but the ball didn't quite fall for the striker and McCormick was able to gather.
Power cut in from the left, ran past two defenders and forced McCormick to save at his near post on 78 minutes while Plymouth substitute Zak Ansah shot wide from close range at the other end.
However, Rovers' survival hopes were dealt a massive blow on 81 minutes. With Tranmere throwing men forward, the hosts hit on the counter attack and Alessandra fired past Fon Williams from 15 yards.
Odejayi pulled a goal back from close range from Donnelly's right wing cross on 89 minutes but it was too little to rescue Rovers' season.
Plymouth: McCormick, Mellor, Nelson, Alessandra (Banton 87), Reuben Reid (Ansah 66), Blizzard (Harvey 87), Holmes-Dennis, McHugh, Bobby Reid, O'Connor, Hartley. Substitutes: Bittner, Norburn, Jones, Brunt.
Tranmere: Fon Williams, Ihiekwe, Dugdale, Donacien, Hill (Hume 66), Laird (Green 59), Power, Jennings, Koumas, Donnelly, Odejayi. Substitutes: Ramsbottom, Gumbs, Kirby, Molyneux, Davies.
Referee: Gavin Ward
Yellow cards: Dugdale (18), O'Connor (74), Power (85)