Aim: To investigate the effects of rat Erythropoietin (Epo) on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods: Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups: the first group was given intratesticular injections of pCAGGSEpo (pCAGGS-Epo group), the second group was given intratesticular injections of pCAGGS (pCAGGS group), and the third group were given intratesticular injections of phosphate-buffered saline (PBS group). At the same time,square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells (G/S ratio) was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results: The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was (0.85 ± 0.08) g and (0.83 ± 0.03) g, respectively, at week 1 (P = 0.788) and (0.62 ± 0.06) g and (0.52± 0.02) g, respectively, at week 2 (P = 0.047). At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group.The G/S ratio was 23.27 ± 6,80 vs. 18.63 ± 5.30 at week 1 (P = 0.0078) and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2(P = 0.1471), respectively. Conclusion: The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.