Spermatozoa produced from spermatogonial stem cells (SSCs) are the vehicle by which genes of a male are passed to the next generation. A single SSC has the ability to self-renew and produce thousands of spermatozoa; therefore, it is an ideal target for genetic modification to efficiently generate transgenic animals in mammalian species. Rats are an important model organism for biological research; however, gene function studies have been difficult because of a limited ability to generate transgenic animals. Transgenic rat production through SSCs offers a means to overcome this obstacle. Because SSCs divide slowly both in vivo and in vitro, lentiviral vectors may be an ideal method for introducing stable genetic modification. Using a lentiviral vector, an enhanced green fluorescent protein (eGFP) transgene was introduced into the genome of cultured rat SSCs, which were microinjected into testes of immunodeficient mice to assess transduction efficiency. Approximately 40% of rat SSCs exposed to the lentiviral vector overnight carried the eGFP transgene and generated colonies of spermatogenesis. When transduced SSCs were transplanted into recipient rat testes, in which endogenous germ cells had been decreased but not eliminated by busulfan treatment, approximately 6% of offspring were transgenic. The transgene was stably integrated into the donor SSC genome and transmitted to and expressed by progeny in subsequent generations. Thus, lentiviral transduction of SSCs followed by transplantation is an effective means for generating transgenic rats through the male germline, and this approach may be applicable to other species in which existing methods are inadequate or not applicable.