Objective To generate Rtn4-A/B knockout mouse model and to explore the biological function of the Rtn4-B gene. Methods The targeting construct for inactivating Rtn4-A/B gene was prepared by bacterial artificial chromosome (BAC). The vector was linearized and electroporated into 129SvEv mouse embryonic stem cells (ES cells). Then the Rtn4-A/B knockout ES cells were microinjected into blastula of C57BL/6J mice after superovulation. F1 hybrid mice were bred to obtain mouse aggregation chimeras, and were identified by PCR amplification of tail genomic DNA. Results Fourteen clones of gene-targeted ES cells were identified after gene knockout and five male chimeras with a higher than 50 chimeric ratio were produced after microinjection into the blastula. Finally four Rtn4-A/B hybrid mice were obtained. Conclusion A Rtn4-A/B deficient mouse strain has been successfully generated by homologous recombination using genetically modified ES cells.