Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 Å resolution, SpnG bound to TDP at 1.86 Å resolution, and SpnG bound to AGL at 1.70 Å resolution. The SpnG–TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG–AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal the features that allow SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction.