Bafilomycin A1 (BAF A1) is a macrolide antibiotic that, in addition to its antibacterial activity, can induce apoptosis of cancer cells. Glycosylation plays an important role in the modification of antibiotics as it can improve their bioactivity. However, glycosylation of BAF A1 has not been previously reported. Bacillus licheniformis glycosyltransferase (GTs) Bl-YjiC and Bacillus subtilis GTs Bs-YjiC were expressed successfully in a heterologous manner. The glycosylation of BAF A1 with UDP-glucose, UDP-galactose or UDP-N-acetylglucosamine was catalyzed using the enzymes Bl-YjiC and Bs-YjiC. Our results demonstrated that Bl-YjiC can only utilize UDP-glucose as the donor, while Bs-YjiC can utilize all three glycosyl donors. The glycosylation site was demonstrated by MS/MS to be the hydroxyl group at the C21 position of BAF A1. The anti-proliferative effects of glucosyl BAF A1 (BAF-Glc) on HeLa cells indicate that this novel antibiotic is superior to BAF A1. The IC50 for BAF-Glc was determined to be 5.47 mu M. Here, we report the production of glycosylated BAF A1 for the first time, and we show that the produced BAF-Glc exhibited better anticancer activity than BAF A1. This work provides theoretical and experimental support for the development of novel anticancer bafilomycins.