Abstract The post-PKS (polyketide synthase) modification reactions, including the hydroxylation step catalyzed by cytochrome P450 monooxygenases, are often crucial to the structural diversity and biological potency of the macrolide polyketides. In this study, we describe a strategy for enhancing the productivity of a set of desired hydroxylated macrolides. In a liquid culture of Streptomyces venezuelae, the intermediate macrolides, YC-17 and narbomycin, accumulate, and a small amount of hydroxylated compounds, namely neomethymycin and pikromycin, is produced. The improved generation of hydroxylated polyketides mediated by the PikC cytochrome hydroxylase from S. venezuelae was accomplished via the overexpression of the pikC gene, the supplementation of ferrous sulfate into the liquid medium, and feeding with the aglycones, 10-deoxymethynolide and narbonolide. In particular, this enhancement of production was achieved with a considerable reduction in culture time. In a liquid culture of a mutant strain (YJ029) that overexpresses the pikC gene, the bioconversion of the 12-membered ring macrolide YC-17 to methymycin and neomethymycin increased by approximately three-fold as compared to that of wild-type S. venezuelae. In the case of the 14-membered ring macrolide, narbomycin, bioconversion to pikromycin increased by approximately five-fold. In addition, the addition of ferrous sulfate and the feeding of aglycones into the medium resulted in a significantly higher generation of the desired hydroxylated macrolides.