The concurrent occurrence of summer heat stress and the non-breeding season has significantly impaired the semen quality of rams. Currently, there exists no straightforward and efficient method to address this issue. In this study, we demonstrate that two consecutive administrations of melatonin implants significantly mitigate the adverse effects of summer heat stress and the non-reproductive season on rams. Our findings indicate that implantation of exogenous melatonin enhances semen quality by improving sperm DNA integrity, mitochondrial integrity, and decreasing the proportion of abnormal sperm, as compared to control rams. This improvement is ascribed to the alleviation of oxidative stress and the optimized regulation of endocrine hormone levels in both serum and seminal plasma. Further exploration of the regulatory mechanism reveals that melatonin can also influence the tryptophan metabolism pathway. Additionally, our study revealed that certain indices and metabolites are strongly correlated with semen quality and can potentially serve as indicators for research aimed at improving semen quality. Notably, this is the first time that differences in tryptophan metabolites between serum and seminal plasma have been elucidated. All the above information suggests that melatonin implantation can protect sperm from heat stress by optimizing the blood and semen microenvironment.