Spinosyn A is a polyketide-derived macrolide produced by Saccharopolyspora spinosa and is an active ingredient in several commercial insecticides. It is glycosylated by a tri-O-methylated rhamnose at C-9 and a forosamine at C-17. Previous studies indicated that the rhamnose methyltransferases are encoded by the spnH, spnl, and spnK genes. To verify the functions of these methyltransferases and to study how they are coordinated to achieve the desired level of methylation of rhamnose, we studied the catalytic properties of the spnH, spnl, and spnK gene products and validated their roles in the permethylation process of spinosyn A. Our data reported herein firmly established that SpnH, Spnl, and SpnK are the respective rhamnose 4'-, 2'-, and 3'-O-methyltransferase. Investigation of the order of the methylation events revealed that only one route catalyzed by Spnl, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety. Moreover, the completion of rhamnose permethylation is likely achieved by the proper control of the expression levels of the methyltransferase genes involved. These results set the stage for future exploitation of the spinosyn biosynthetic pathway to produce targeted spinosyn derivatives and, perhaps, new analogues.