FK506, structurally similar to FK520 and rapamycin, is an α-keto amide bonding-containing, macrolide natural product that exhibits potent immunosuppressive activity and moderate antifungal activity. FK506 biosynthesis requires a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) system to construct the skeleton of the macrolide. The mechanism for tailoring this macrolide to furnish FK506 remains poorly understood. In this study, we report a maturation paradigm common for FK506, FK520, and rapamycin, by characterizing two conserved regiospecific, post-PKS-NRPS modifications in an FK506-producing Streptomyces tsukubaensis strain. A cytochrome P450 protein, FkbD, catalyzes a less common, four-electron oxidation at C-9 to give a rarely found α-keto amide group, whereas a methyltransferase, FkbM, is responsible for O-methylation at C-31 to afford a methoxy group. Both FkbD and FkbM are highly tolerant in their substrate choice; therefore, the order of FkbD- and FkbM-catalyzed reactions is interchangeable in the FK506 biosynthetic pathway. Inactivation of fkbD produced a new intermediate, 9-deoxo-FK506, which displayed antifungal activity lower than that of FK506. Taking previously reported bioassay results regarding the intermediates 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 into account, it is clear that the modifications catalyzed by FkbD and FkbM are of importance to reach the full biological activity of FK506 by forming a key structure motif that is necessary for interaction of the molecule with the receptor and, subsequently, the downstream intracellular responses.