Prime Minister Edouard Philippe took temporary control of the interior ministry while Macron began searching for a replacement for 71-year-old Collomb, who was one of the first politicians to back him for president.
The fate of Collomb, nicknamed "France's top cop" because his ministry is in charge of security and immigration, has thrown the government into flux.
Macron initially refused his resignation on Monday, but on Tuesday night gave into Collomb's request to be allowed to run again for his former job as mayor of the eastern city of Lyon.
The Liberation newspaper described the back-and-forth as "extraordinary dilly-dallying which seems more like something from a music-hall than government politics".
Collomb's departure adds to the woes of the France's centrist leader, who is battling record low approval ratings after 17 months in power.
It comes just weeks after popular environment minister Nicolas Hulot resigned live on radio without warning Macron, saying he felt "all alone" in the government on green issues.
A stony-faced Philippe vowed at a handover ceremony Wednesday to "maintain the highest level of security for French people" while in charge of the interior ministry.