BACKGROUND: Heat stress (HS) commonly occurring in summer has gradually become a factor threatening the reproductive performance of male dairy goats by reducing their fecundity. Despite the melatonin is applied to relieve HS, it is still unclear whether melatonin protects against reproductive damage induced by HS in dairy goats and how it works. The purpose of the present study is to evaluate the role of melatonin in alleviating HS-induced spermatogenesis dysfunction in male dairy goats and further explore its mechanism. RESULTS: HS impaired spermatogenesis, sperm formation in the testes, and sperm maturation in the epididymis of dairy goats, resulting in decreased sperm quality. Melatonin rescued the decrease of sperm quality induced by HS via decreasing inflammatory and oxidative stress levels in testicular tissue and enhancing intercellular barrier function within the testes. Amplicon-based microbiota analysis revealed that despite gut microbiota differences between melatonin-treated dairy goats and NC dairy goats to some extent, melatonin administration tends to return the gut microbiota of male dairy goats under HS to the levels of natural control dairy goats. To explore whether the protective role of melatonin in sperm quality is mediated by regulating gut microbiota, fecal microbiota of HS dairy goats with or without melatonin treatment were transferred to HS mice, respectively. We found HS mice that had received fecal bacteria of HS dairy goats experienced serious testicular injury and dyszoospermia, while this phenomenon was ameliorated in HS mice that had received fecal bacteria of dairy goats treated with melatonin, indicating melatonin alleviates HS-induced spermatogenic damage in a microbiota dependent manner. We further found that the testicular tissue of both HS dairy goats and mice transplanted with HS dairy goat feces produced large amounts of arachidonic acid (AA)-related metabolites, which were closely associated with semen quality. Consistently, supplementation with AA has been shown to elevate the levels of inflammation and oxidative stress in the testicular tissue of mice, disrupting intercellular connections and ultimately leading to spermatogenic disorders. CONCLUSION: This study has revealed that melatonin can effectively alleviate spermatogenic disorders in dairy goats caused by HS. This beneficial effect was primarily achieved through the modulation of gut microbiota, which subsequently inhibited the excessive synthesis of AA in testicular tissue. These discoveries are of great significance for preventing or improving the decline in male livestock reproductive performance caused by HS, enhancing the reproductive efficiency of elite male breeds, and ultimately improving the production efficiency of animal husbandry. Video Abstract.