The former Falkirk boss had been in charge since August 2018 and guided the West Lothian club to fifth place in the Premiership last season.
Livingston are currently sitting 10th out of 12 in the top flight, with 12 points from 15 matches and Holt told the club website: "It is with great regret that I have made the difficult decision to resign as manager of Livingston FC.
"This is not a decision I have taken lightly but feel it is the right time for me to step away. 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 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.”
Livingston have confirmed that David Martindale and Tony Caig will take charge on an interim basis, with their next match coming on Saturday when Ayr United visit in the Betfred Cup.