The molecular basis of most human male infertility arising from spermatogenesis disruption is poorly understood because of a lack of useful investigation systems. To study the roles of the supporting Sertoli cells in mammalian spermatogenesis, we improved an electroporation technique for seminiferous tubules in vivo. Because Sertoli cells barely proliferate in mature testis, linear transgenes are not incorporated into the genome and quickly degrade. However, circular expression vector is stably expressed in Sertoli cells for a long period. By electrotransformation of a complete cDNA, we rescued defective spermatogenesis in infertile Sl^17H/Sl^17H mutant mice with partial dysfunction of stem cell factor in Sertoli cells. Application of this gene transfer system will facilitate both the understanding of spermatogenesis and the development of new gene therapies for human male infertility.