Microorganisms from culture collections and isolates from nature were screened for the ability to catalyze the regioselective glucosylation of pyridoxine (PN) to produce pyridoxine 5'-alpha-D-glucoside (PN-5'-alpha-G) or pyridoxine 4'-alpha-D-glucoside (PN-4'-alpha-G). Transglucosylation activity specific to 5'-position of PN was found in fungi belonging to genera such as Coriolus and Verticillium, and activity at the 4'-position of PN was found in bacteria belonging to genera such as Bacillus and Serratia. From 100 mM PN, intact cells of Verticillium dahliae TPU 4900 produced 42 mM (13.9 mg/mL) PN-5'-alpha-G after 70 h of reaction. Intact cells of Bacillus cereus TPU 5504 produced 33 mM (10.9 mg/mL) PN-4'-alpha-G after 19 h of reaction. The selectivities for 5'- and 4'-positions were 80% and 90%, respectively.