The present study aimed to identify whether vitamin E and C and their combination in basal freezing medium improved the viability of postthaw transfected spermatogonial stem cells (SSCs). For this purpose, SSCs were harvested by enzymatic digestion twice. After enrichment and culture on Sertoli cell feeder layer, SSCs were characterized by using alkaline phosphatase staining and expression of oct-4 and c-kit genes as specific stem cell markers. As to the transfection of SSCs, different concentrations of DNA (0.2, 0.4, and 0.8 μg) and turbofect (0.4 and 0.8 μL) in 100 μL of medium were studied. The results showed that cryopreservation of SSCs in the presence of 25 μg/mL vitamin E and 10 μg/mL vitamin C could increase cell viability and expression of antiapoptotic genes. The best combination of DNA and turbofect for transfection of SSCs in 100 μL of medium was 0.8 μg and 0.4 μL, respectively. However, SSCs indicated lower transfection efficiency compared with Sertoli cells (~30% vs. 70%, respectively). Cryopreservation with the addition of vitamin E and C in the basal freezing medium could increase cell viability of transfected SSCs as well. The findings of this study also suggest the need for further research regarding improvement of the transfection efficiency of SSCs.