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Objective: We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irradiated testes.Materials and Methods: In this experimental research, the right testes from adult mice (n=25) were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine (BrdU)-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells (control 1), fresh cells co-cultured with Sertoli cells (control 2), the frozen-thawed cells (experimental 1) and frozen-thawed cells co-cultured with Sertoli cells (experimental 2). The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation.Results: The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient’s testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups (p≤0.001).Conclusion: Our results demonstrated that spermatogonial stem cells in the colonies could result sperm production in the recipient’s testes after autologous transplantation.