Objective To improve the current method of spermatogonial cells transplantation, and make the new method become more operative and easy to carry out. Methods The spermatogonial cells in rC57BL6/tg14 (act-EGFO-Osb Y01) mice that expresse GFP protein were collected as the donor cells, and by using a modified syringe, and then were injected into the seminiferous tubules of pretreated wild type GFP-null mice through testicular efferent duct. The transplantation outcome was evaluated by trypan blue stainting and fluorescent microscopic examination and pathohistological analysis of transplanted testis. Results The transplanted testis of recipient mice showed green fluorescence signal, and the signal of seminiferous tubules was found significantly higher than that of the surrounding tissues. The transplanted GFP-positive cells generated colonies and spermatogenesis but pretreated wild type GFP-null mice were not found. Conclusion The expressed GFP protein spermatogonial cells in rC57BL6/tg14 mice were successfully transplanted in the wild type GFP-null recipient mice, fourthermore the improved transplantation method simplified the triditional one and achieved the same transplantation results.