The microbial transformation of a series of tetrahydroprotoberberines (THPBs, 1-5) by Gliocladium deliquescens NRRL1086 was investigated. In this research, the novel glycosylation of tetrahydroberberrubine (1) was observed with fast rate and high regio- and enantio-selectivity. One pair of unique enantiomorphic alkaloidal glycosides T-1 and T-2 was isolated and their structures were elucidated unambiguously by HR-MS, CD, 1D and 2D NMR spectrum. It is interesting that different amounts of glucose in the potato broth medium could influence the ratio of T-1 and T-2; in the 1.5% glucose medium, the ratio was about 15:1 and the yield of the S-form product T-1 may reach the theoretical maximum yield of about 50% which could provide one practical method to prepare the enantiomerically pure product and one alternative resolution method of tetrahydroberberrubine. The preliminary enzymatic research by using sodium dodecyl sulfate (SDS) and imidazole as glycosyltransferase and glycosidase inhibitors revealed that glycosyltransferase may contribute to glycosylation process. This is the first successful approach to glycosylation of tetrahydroprotoberberines.