For the development of a practical process producing D-homoserine from DL-homoserine, microbial optical resolution was investigated. A bacterial strain 2-3 which can enantioselectively assimilate the L-enantiomer from DL-homoserine was isolated, and identified as Arthrobacter nicotinovorans. The strain 2-3 grew on a medium containing 5% (w/v) of DL-homoserine hydrobromide and exhausted L-enantiomer from the culture to leave D-enantiomer, which was recovered with enantiomeric excess of over 99.9%. The present method is simple and suitable for practical use.