Abstract： Background Evidence for the importance of genetic factors in male infertility is accumulating. This study was designed to identify a novel testis-specific gene related to spermatogenesis by a new strategy of digital differential display (DDD).Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene. Multi-tissue RT-PCR was performed to analyse its tissue-specific expression. The full-length cDNA of the new gene was obtained using the BLAST program. Sequencing was performed and the result was analysed. Semi-quantitative RT-PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene. The sequence of the opening reading frame was integrated into the pQE-30 vector expressed in Escherichia coil strain M15(pREP4). With IPTG induction, the target protein was detected.Conclusions DDD can be confirmed by SPATA12 as a novel computational biology-based approach for identification of the testis-specific expression genes. SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis. Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein.