Melatonin receptors (MT1 and MT2) were shown to regulate proper functioning of reproductive system, especially in seasonally breeding animals. European bison is a unique endangered seasonal breeder and knowledge of the molecular mechanisms of its reproduction is crucial for the survival of the species. The aim of this study was to assess gene expression, protein synthesis and immunohistochemical localization of MT1 and MT2 receptors in testicular and spermatic cord vessels tissues collected in pre-rut (June) and post-rut (December) seasons from adult male European bisons in Bialowieza National Park. We confirmed the highest expression of MT1 and MT2 mRNA and protein levels in testis in December, while in spermatic cord gene expression was also highest in December, but protein amounts were comparable in both analyzed periods. Furthermore, immunohistochemical staining revealed the same amount of both receptors in arteries and veins of spermatic cord in both periods and increased amounts in December in Leydig, Sertoli and germ cells. The high level of testicular melatonin in December confirms the inhibition of spermatogenesis and increased anti-oxidant and anti-inflammatory protection. In spermatic cord vessels, it may prevent from age-related changes due to the overexploitation and ensure a constant temperature regardless of changing environmental conditions. This knowledge can contribute to finding a solution of problems associated with male infertility in general and also further explore the mechanisms regulating the proper functions of the male reproductive system.