Mitomycins are a family of naturally occurring, potent alkylating agents in which the C member has been clinically used for cancer chemotherapy for over 5 decades. In Streptomyces caespitosus, mitomycins are derived from an N-glycoside composed of a 3-amino-5-hydroxybenzoic acid (AHBA) unit and a d-glucosamine (GlcN) unit; however, how this N-glycoside is formed and rearranged to a mitosane, for example, the compact polycyclic ring system of mitomycin C, remains elusive. Benefiting from the development of a method used to trace the mitomycin intermediates that accumulate on an acyl carrier protein (ACP), we here dissect the enzymatic steps for AHBA-GlcN formation and processing to underlie the mitosane structure. Following the N-glycosylation of AHBA with activated N-acetyl-GlcN, deacetylation occurs on ACP to provide AHBA-GlcN. Then, the sugar portion of this N-glycoside is transformed into a linear aminodiol that terminates with an epoxyethane, yielding an ACP-channeled intermediate that is ready for mitosane formation through crosslinking between the AHBA and linearized sugar units. This transformation is unusual and relies on the functional association of a dihydronicotinamide adenine dinucleotide (phosphate)-dependent protein with a radical S-adenosyl-l-methionine protein. Characterization of these ACP-based enzymatic steps for AHBA-GlcN formation and processing sheds light on the poorly understood biosynthetic pathway of mitomycins.