The 66-year-old former Manchester City boss arrived at the Keepmoat Stadium to assist boss Paul Dickov in June 2013.
Horton told the club’s official website: “It’s been a thoroughly enjoyable two years here and I leave knowing that the club and the team are in a better place.
“Paul is working hard to attract more new players here and to build a team that can push for a play-off place and ultimately get out of this division.
“The team and the squad will look different this year, it is being built around players that want to fight and to play as one unit, not individuals. That’s what the fans, and Paul deserve.”
Horton started out in management at Hull in 1984 and has had spells in charge at Oxford, Manchester City, Huddersfield, Brighton, Port Vale and Macclesfield (twice).
Dickov added: “I would like to thank Brian for his time here, he has been a great guy to have around and to work with.
“I have known Brian for years and over that time, including his two years here I have been able to lean on him for his experience and unbelievable knowledge of the game.
“The man is a font of all knowledge when it comes to football and we all have the upmost respect for him. It’s been an absolute pleasure having him around and we wish him well.”