To increase the bioavailability of berberine, three new 9-O-glycosyl-berberine derivatives, 9-O-glucosyl-(4a), 9-O-arabinosyl-(4b), and 9-O-erythrol-(4c) were obtained and confirmed by UV, 1 HNMR, 13CNMR, and MS. The pharmacokinetic profiles of these synthetic compounds have been evaluated compared with berberine (1) and 9-O-alkyl-berberine (5) derivatives, which showed that maximum concentration (Cmax) and area under concentration–time curve (AUC) of 9-O-glycosyl-berberine increased dramatically. The results indicated that hydrophilic modification could significantly improve the bioavailability of berberine; 9-O-glycosyl-berberine might be a promising prodrug.