The spermatid-specific gene, sp3111, is a new member of the four-transmembrane gene family. However, its reproductive biological function remains elusive. The aim of this study was to establish an sp3111-knockdown male mouse model for further studying the effect of suppression of sp3111 gene expression on its reproductive functions. Recombinant vectors of pSUPER-sp3111-shRNA, containing the transcribed functional small hairpin RNA sequence against different regions of mouse sp3111 mRNA, were constructed and identified. Then, the linearized recombinant vectors were injected into mature mouse testes and transfected to spermatogonial cells by electroporation to generate sp3111 transgenic RNAi mice. These electroporated male mice were mated with wild-type females 30 days after electroporation and their progenies were examined both by fluorescence microscopy and PCR. It was found that the suppression efficiency of RNAi on the testis expression of sp3111 mRNA in vivo was more than 30% and could be transmitted stably to F3 generation, and compared with the wild-type male mice, the mean number of offspring of the sp3111 RNAi male mice was reduced from 11+1.3 to 6+1.4. In conclusion, the intra-testicular microinjection technique could also be used to develop the transgenic RNAi mice, and sp3111 transgenic RNAi mouse model may provide the possibility to further study the sp3111 gene reproduction function in vivo.