Various methods are currently under investigation to preserve fertility in males treated with high-dose chemotherapy and radiation for malignant and nonmalignant disorders. Human umbilical cord mesenchymal stem cells (HUC-MSCs), which possess potent immunosuppressive function and secrete various cytokines and growth factors, have the potential clinical applications. As a potential alternative, we investigate whether injection of HUC-MSCs into the interstitial compartment of the testes to promote spermatogenic regeneration efficiently. HUC-MSCs were isolated from different sources of umbilical cords and injected into the interstitial space of one testis from 10 busulfan-treated mice (saline and HEK293 cells injections were performed in a separate set of mice) and the other testis remained uninjected. Three weeks after MSCs injection, Relative quantitative reverse transcription polymerase chain reaction was used to identify the expression of 10 of germ cell associated, which are all related to meiosis, demonstrated higher levels of spermatogenic gene expression (2-8 fold) in HUC-MSCs injected testes compared to the contralateral uninjected testes (five mice). Protein levels for germ cell-specific genes, miwi, vasa and synaptonemal complex protein (Scp3) were also higher in MSC-treated testes compared to injected controls 3 weeks after treatment. However, no different expression was detected in saline water and HEK293 cells injection control group. We have demonstrated HUC-MSCs could affect mouse germ cell-specific genes expression. The results also provide a possibility that the transplanted HUC-MSCs may promote the recovery of spermatogenesis. This study provides further evidence for preclinical therapeutic effects of HUC-MSCs, and explores a new approach to the treatment of azoospermia.