The 47-year-old had previously stated he would need to consider his role following a 1-0 home defeat by St Mirren on Saturday, which left the Lions third bottom of the Scottish Premiership.
Holt’s team have only won three of their 15 league games this season
In a statement on the club’s official website, Holt, who took over at Livi following Kenny Miller’s brief spell in charge in August 2018, said: he had made his decision with “great regret”.
“This is not a decision that I have taken lightly but feel it is the right time for me to step away,” he said.
“I am proud to say the work we have done over the past two years has resulted in great success both on and off the pitch.
“The progression the club has seen has been remarkable to be part of and last season’s fifth-place finish was one of many highlights.
“I would like to place on record my sincere gratitude to the club, fans and players for all their support during my tenure.
“I wish nothing but the best to everyone connected to the club for the future and hope there is a great deal more success to come.”
Former Scotland international Holt was in charge at Falkirk between 2013-14 before leaving to become first team coach at Norwich City.
Livingston’s head of football operations, David Martindale, who shared the dugout with Holt, said: “Gary has been tremendous for the club over the past few seasons and we have all enjoyed working with him on and off the park.
“It’s very rare in football these days that all parties come out the other end with their relationships firmly intact however, I can honestly say this is the case with Gary’s resignation.
“It’s been a tough season and we are probably a victim of our own success in comparison to last season, which ultimately raised everyone’s expectations including players along with all staff at the club.
“So, when we don’t quite hit those heights again it can feel like something needs to change and I totally understand Gary’s thought process.
“Gary can leave Livingston knowing he has left us in a better place than when he arrived.”
Martindale and Tony Caig will take interim charge ahead of Saturday’s Betfred Cup encounter with Ayr.