Rate acceleration of O-glycosylation had been observed in p-xylene under frozen conditions, when thioglycosides were activated by methyl trifluoromethane sulfonate. Curiously, significant perturbation of stereoselectivity was observed. Effects of various factors, such as solvent, concentration, anomeric configuration and protective groups of the donor, were systematically examined to clarify the mechanistic implications of stereoselectivity on glycosylation under frozen system. Our study revealed that the stereoselectivity was affected by concentration both in liquid as well as in frozen conditions, indicating that rate acceleration effect in frozen solvent was caused by highly concentrated environments.