Horton, 63, returned for a second spell in charge at the Moss Rose last month after Gary Simpson was sacked on the back of 14 league matches without victory.
However, the former Manchester City and Port Vale chief was unable to bring about a change in fortunes and eight more winless games garnered only two points, culminating in Saturday's relegation-sealing 2-0 home loss to Burton.
Glyn Chamberlain, who served as assistant to both Simpson and Horton, will take charge for the Silkmen's final game of the season at Southend and vice chairman Andy Scott insists work is already underway to put a permanent successor in place after concluding Horton was not the man to mastermind a return to the Football League.
"We need a different set of skills to be competitive in the Conference," he told Macclesfield's official website. "Our favoured candidate will be someone who knows the game well at that level.
"We have an excellent set up behind the scenes, which we think will offer an attractive proposition to an ambitious manager. We have some targets in mind and we hope to be able to make an early permanent appointment."