Methotrexate (MET) is one of the most important chemotherapy agents used against various tumors and cancer diseases. One of the critical side effects of MET is inducing male infertility. The current study aimed to investigate Sertoli cell culture-conditioned medium (SCM) recovery effects on MET-induced conditions in rats. 30 mature male Wistar rats were randomly divided into 3 groups (n = 10). In the first group, rats received normal saline intraperitoneally. In the second group, animals received MET (10 mg/kg; intraperitoneally) once a week for 2 wk. The rats in the third group (MET+SCM) received MET and a single injection of SCM for 56 days post-MET administration. 56 days later, serum, epididymis, and testicular tissue samples were collected, and the animals were euthanized. Sperm parameters, serum levels of luteinizing hormone, follicle-stimulating hormone, and testosterone were examined. The testicular tissues were stained using hematoxylin and eosin solution, and histopathological changes were analyzed. The MET-induced condition resulted in significant pathological changes in the testis, decreased hormone levels, and downregulated sperm parameters. However, SCM injection improved hormonal levels, testicular changes, and sperm parameters. It can be concluded that a single intra-testicular SCM injection accelerates male reproductive system recovery post-MET treatment.