The Quakers lost 1-0 at Curzon Ashton yesterday which leaves them in 12th place in the Vanarama National League North.
A statement by the club's board of directors reads: "Darlington Football Club today regretfully confirm the resignation of Martin Gray as our manager.
"After discussions with club directors John Tempest and David Johnston this morning, Martin has made it clear that he believes he has taken the club as far as it can go, given the current club structure and funding.
"We would like to put on record our thanks to Martin for five fantastic years, during which he has guided the club from the Northern League to National League North, winning three promotions along the way. We wish him well in his future career.
"Brian Atkinson and Sean Gregan will take care of first team affairs until the end of the season. They have both met the directors, and have expressed a hunger and desire to take the club forward. They have impressed the board with their vision on how they would bring fresh ideas and impetus to the club.
"We wish Brian and Sean the best of luck in their new roles."