The hypoxic microenvironment of cryptorchidism is an important factor in the impairment and fibrosis of Sertoli cells which result in blood-testis barrier (BTB) destruction and spermatogenesis loss. Recent studies have shown that melatonin, a well-known pineal hormone exerts beneficial effects against pathological fibrosis in a various of organs. However, it is still unknown whether melatonin can regulate hypoxia-induced fibrosis of Sertoli cells. In this study we evaluate melatonin levels, and its synthesizing enzymes, AANAT and HIOMT expression patterns in canine cryptorchidism and contralateral normal testis. Results show abdominal testes presented low melatonin levels and AANAT and HIOMT expression compared with testes located in the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2) and found that melatonin inhibited the EMT markers expression and ECM production as well as Hif-1 alpha expression of Sertoli cells in a dose-dependent manner. Furthermore, use of Lificiguat (synonyms YC-1, Hif-1 alpha inhibitor) to interfere with the Hif-1 alpha pathway showed a similar effect with melatonin suppression of the fibrosis in Sertoli cells. The results indicate that melatonin supplementation can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with regulating the inhibition of Hif-1 alpha signaling.