In order to isolate a microorganism having preferential degradation of D-carnitine from DL-carnitine, a bacterium assimilating D-carnitine as a sole carbon and energy source was isolated from soil by enrichment culture and partially identified as Enterobacter sp. Also, a mutant having lessened L-carnitine decomposition rates was selected with nitrosoguanidine mutagenesis, which led to decrease the specific activities of carnitine dehydrogenase (7.6-fold) and ${eta}$-hydroxybutyrate dehydrogenase (9.5-fold) as compared to the wild strain. Meanwhile, optimal culture conditions for optical resolution of DL-carnitine were investigated. Under optimal conditions, 3.53 g/l L-carnitine was obtained from 20 g/l DL-carnitine, which corresponded to 35.3% L-carnitine yield and 97.9% optical purity.