Pseudomonas sp. PA09 was isolated from farm soil and used for the optical resolution of D-pipecolic acid from DL-popecolic acid. The strain PA09 consumed L-pipecolic acid preferentially as the sole carbon and energy source, thus accumulating D-pipecolic acid in the culture broth. Optimization to improve the enantiomeric excess and yield was performed. The time course experiment showed that the strain OP09 consumed L-pipecolic acid almost to completion after 35h of cultivation, and the enantiomeric excess and the yield (% of residual D-pipecolic acid) were 99.8 and 96.0%, respectively.