Stags slipped to a first Papa Johns Trophy loss of the season as first half goals from Otis Khan and Danilo Orsi saw Grimsby win 2-0 at Blundell Park.
In the previous game of the group stage, Mansfield beat Manchester City under-21s 3-2 in a thrilling match at One Call Stadium but this was a less exhilarating spectacle.
Eight changes were made from the side which beat Gillingham 2-0 on Saturday in Sky Bet League Two, with Kellan Gordon, Stephen McLaughlin and Anthony Hartigan surviving in the starting XI.
James Perch captained the team whilst Adam Collin, the academy goalkeeping coach, was registered in the squad and named as substitute goalkeeper.
Former Mansfield man Otis Khan made his debut for Grimsby.
And it was the diminutive midfielder who almost had an early opener inside the fourth minute when he broke clear of the Stags' defence, but Mansfield goalkeeper Scott Flinders did well to narrow the angle and deny the low effort with his legs.
At the other end, Gordon linked up well with Will Swan but the Nottingham Forest loanee was unable to get a clean shot away and the opportunity went amiss.
John-Joe O'Toole came closer to a breakthrough in the 13th minute as his bullet header flew over the bar following Anthony Hartigan's pin-point right-sided free-kick.
But it was Khan who poked Grimsby in front with a quarter-of-an-hour played.
In a similar move to the first, Khan raced away from the defence to get beyond O'Toole and, significantly, Scott Flinders, before slotting home.
Flinders' palms were stung two minute later as he claimed a venomous long-range from Grimsby's Keyendrah Simmonds.
In the 28th minute, Anthony Hartigan required treatment from the physio following a challenge in midfield and was soon replaced by George Maris.
Stags then broke from midfield and almost levelled when Swan latched on to Gale's precise pass inside the box, but directed his low effort narrowly beyond the far post.
The pair combined again, this time on the inside right, though Swan saw his low attempt well held by home goalkeeper Max Crocombe.
John-Joe O'Toole was then shown a yellow card in the 33rd minute for a foul near the halfway line.
Just when Stags looked like they were getting a foothold in the tie, Grimsby doubled their advantage.
Perch and Bowery both attempted to make interventions midway inside the Stags' half, and were undone when Simmonds fed Danilo Orsi who peeled away from his marker to advance and fire low beyond Flinders.
In the final minute of normal time at the end of the half, home 'keeper Crocombe was well behind McLaughlin's well hit left-footed effort as Stags tried in vein to pull one back.
And it was the last meaningful act of the night for McLaughlin as he was replaced by Elliott Hewitt at the break.
Mansfield restarted in a 4-3-3 fashion, with Bowery moved up front to the left of a three-pronged attack.
Two minutes into the half, Crocombe was equal to Perch's effort on the turn following a Stags' corner.
Mansfield entered the second period with new-found gusto and on 49 minutes, Bowery's powerful effort from the edge of the box was blocked after Swan's square pass.
Moments later, the Stags defender-cum-striker saw his curling effort tipped away by Crocombe as Mansfield upped the ante.
Just shy of the hour, Max Clifton blasted over into an empty stand in Grimsby's first noteworthy chance of the half.
The hosts looked to get back into their groove and were denied by an expert save by Flinders from a corner, who clawed away a Mariners' attempt inside a crowded penalty box.
On 63 minutes, Swan did superbly to hold up a high falling ball inside the box, and after laying it back to Gale, the former Long Eaton striker was unable to keep his shot down.
Gordon went down under a challenge with 22 minutes remaining and was replaced by Jason Law.
On 73, George Lapslie and Riley Harbottle were introduced for Stephen Quinn and Kieran Wallace.
Two minutes later, Swan was a stride away from connecting with a lovely throughball from Maris as Stags sought to reduce the arrears.
Grimsby should have wrapped it up when substitute Aribim Pepple raced down the middle, going one-on-one with Flinders, only to steer his effort wide.
In front of 141 traveling Stags' fans, Crocombe went untested as Harbottle headed over from a corner.
With eight minutes to go and Mansfield pushing for an equaliser, Gale nodded a floating header beyond the far post.
It was the last opportunity of note as Stags were unable to find the net.
GRIMSBY: Crocombe, Efete, Maher, Pearson, Amos, Khan (Pepple 46), Clifton (holohan 88), Khouri, Maguire-Drew (Richardson 79), Simmonds (Wearne 46), Orsi (Glennon 88)
UNUSED SUBS: Battersby, Smith
MANSFIELD: Flinders, Gordon (Law 69), O'Toole, Bowery, Perch, McLaughlin (Hewitt 46), Hartigan (Maris 30), Wallace (Harbottle 73), Quinn (Quinn 73) , Gale, Swan
UNUSED SUBS: Collin, Hawkins
REFEREE: Robert Madden
ATTENDANCE: 912 (144 Stags' fans)
