<jats:title>Abstract</jats:title><jats:p>Sch47554 and Sch47555 are antifungal compounds from<jats:italic>Streptomyces</jats:italic>sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with<jats:italic>schS7</jats:italic>in<jats:italic>Streptomyces lividans</jats:italic>K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a<jats:sc>d</jats:sc>‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an<jats:sc>l</jats:sc>‐aculose or<jats:sc>l</jats:sc>‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an<jats:sc>l</jats:sc>‐aculose moiety to 3‐OH of the angucycline core.