Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.