The League Two outfit are yet to taste victory in 2012 and Saturday's defeat to fellow strugglers Dagenham stretched their winless run to 16 matches in all competitions.
Simpson took over as manager at the Moss Rose two years ago following the death of Keith Alexander, who he worked under as assistant, and he leaves with the Silkmen outside the League Two relegation zone on goal difference.
A statement on Macclesfield's official website read: "Following a sequence of results that has left Macclesfield Town without a win in 2012, the club's board has asked Gary Simpson to step down from his role as manager."
Chairman Mike Rance added: "It is with great regret that we've had to make this decision.
"We've been through an immense amount together over the last two years and we owe Gary a real debt of gratitude for the dignity and commitment he has shown through a difficult time."