Objective To compare the effects of different transplantation sites on the outcome of testicular grafts in mice, and to provide a basis for development and application of this technique in relevant research. Method 5-day old and 4-week old SPF male C57BL/6J mice were used in this study. Three groups of testicular transplantation, i.e. dorsal subcutaneous transplantation (5 mice, 40 testes), transplantation inside the testicular tunica albuginea (6 mice, 12 testes), and were subrenal capsule transplantation (10 mice,15 testes) groups were set up for evaluating the effect of transplantation site on the outcomes in mice. Sham operation (4 mice) and castration (4 mice) groups were also used in this study. The mice were sacrificed at 8 weeks after transplantation and the transplanted testes were collected for analysis of their weight, transplant survival, weight gain, and germ cell differentiation. Results There were significant differences of the testicular germ cell differentiation in different transplantation groups. The germ cell differentiation was best in the intra-tunica albuginea transplantation group, and were similar to that in the sham operation group. The germ cell differentiation rate was 100% in the intra-tunica albuginea transplantation group, 29.2% in the subcutaneous transplantation group and 0% in the subrenal capsule transplantation group. Conclusions Transplantation beneath the testicular tunica albuginea is the most favorable site for germ cell differentiation, and dorsal subcutaneous transplantation is an alternative choice. Subrenal capsule transplantation is not appropriate for preservation of male reproductive organs in mice.