Mithun (Bos frontalis) is a unique domestic free range bovine species of North Eastern Hilly (NEH) regions of India. Effect of feed supplementation of Flaxseed oil (FSO) on semen production and its quality profiles, freezability, oxidative stress, apoptotic sperm percentage and subsequently on endocrinological profiles & scrotal and testicular biometrics in different seasons was studied in mithun. The experimental animals were divided into two groups, Gr I: Control (n = 3) and Gr II: Treatment (n = 3; Flaxseed oil @ 150 mL/day). FSO was supplemented through oral drench in the morning hours just before concentrate feeding. A total of 80 semen samples (n = 80; 20 semen samples from each season; each 10 semen samples from control and treatment groups per season) were collected, not more than twice per week in winter, spring, autumn and summer seasons. Semen quality profiles (SQPs) such as volume, sperm concentration, motility (forward progressive and total), motility & velocity profiles by computer assisted sperm analyser (CASA), viability, total sperm abnormality, acrosome integrity, plasma membrane & nuclear abnormality and apoptotic sperm percentage were estimated in fresh semen. Along with SQPs measured in fresh semen, motility in estrus bovine cervical mucus (bovine cervical mucus penetration test; BCMPT) and mitochondrial membrane potential (MMP) by JC-1 stain were determined in the post-thawed semen samples. Biochemical profiles (aspartate aminotransferase; AST, alanine aminotransferase; ALT, total cholesterol; CHO), antioxidant profiles (superoxide dismutase; SOD, catalase; CAT, glutathione; GSH, total antioxidant capacity; TAC) and oxidative stress profile (malondialdehyde; MDA) were estimated in fresh semen whereas AST, ALT, lactate dehydrogenase (LDH), TAC and MDA were estimated in the frozen thawed semen samples. Endocrinological profiles such as follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, cortisol and thyroxin and scrotal circumference (SC) & testicular biometrics were measured in both groups in different seasons. Result revealed a significant (p < 0.05) improvement in motility (total & forward progressive, motility & velocity by CASA and vanguard distance in cervical mucus), viability, intactness of acrosome & plasma membrane, MMP, antioxidant profiles and reduction in total sperm and nuclear abnormalities, reduction in leakage of intracellular enzymes and reduction in oxidative stress profile and reduction of apoptotic sperm percentage were observed in FSO supplemented than in un-supplemented control group accordingly in fresh and post thawed semen samples. Blood FSH, LH, testosterone and thyroxin concentration were significantly (p < 0.05) increased and cortisol concentration was significantly (p < 0.05) decreased in FSO supplemented group than in unsupplemented control group. Similarly, SC and testicular biometrics were increased significantly (p < 0.05) in supplemented than unsupplemented group for different seasons and significantly (p < 0.05) higher in winter and spring than in summer season in the experimental groups. It can be concluded from the study that supplementation of FSO can effectively be utilized to improve the antioxidant profiles, reduction of oxidative stress with cascading beneficial effects on SQPs and fertility status of the mithun bull.