A mutant Xanthomonas maltophilia BT-112 with high α-anomer-selective glycosylation activity was screened by a series of mutation methods including UV light, N-methyl-N-nitro-N-nitroso-guanidine treatment and quick neutron mutation. The α-arbutin titer increased 15-folds compared with the parent strain. The optimal conditions for culture medium and the operational conditions for lab-scale fermenter were investigated. Under optimized conditions, the maximal hydroquinone (HQ) tolerance of cells and yield of α-arbutin were 120 mM and 30.6 g/l, respectively. The molar conversion yield of α-arbutin based on the amount of HQ supplied reached 93.6 %. The product was identified as α-arbutin by (13)C NMR and (1)H NMR analysis. In conclusion, the results in this work provide a one-step and cost-effective method for the large-scale production of α-arbutin.