<jats:title>ABSTRACT</jats:title> <jats:p> Spiramycins are clinically important 16-member macrolide antibiotics produced by <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Streptomyces ambofaciens</jats:named-content> . Biosynthetic studies have established that the earliest lactonic intermediate in spiramycin biosynthesis, the macrolactone platenolide I, is synthesized by a type I modular polyketide synthase (PKS). Platenolide I then undergoes a series of post-PKS tailoring reactions yielding the final products, spiramycins I, II, and III. We recently characterized the post-PKS glycosylation steps of spiramycin biosynthesis in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">S. ambofaciens</jats:named-content> . We showed that three glycosyltransferases, Srm5, Srm29, and Srm38, catalyze the successive attachment of the three carbohydrates mycaminose, forosamine, and mycarose, respectively, with the help of two auxiliary proteins, Srm6 and Srm28. However, the enzymes responsible for the other tailoring steps, namely, the C-19 methyl group oxidation, the C-9 keto group reduction, and the C-3 hydroxyl group acylation, as well as the timing of the post-PKS tailoring reactions, remained to be established. In this study, we show that Srm13, a cytochrome P450, catalyzes the oxidation of the C-19 methyl group into a formyl group and that Srm26 catalyzes the reduction of the C-9 keto group, and we propose a timeline for spiramycin-biosynthetic post-PKS tailoring reactions.