BACKGROUND: The association between antisperm antibodies (ASA) and male (in)fertility is not fully understood, mainly due to contradictory reports. However, there is indication that ASA might affect several male reproductive health aspects warranting further investigation. OBJECTIVE: To investigate the prevalence and impact of ASA on semen quality and its association with male reproductive function. MATERIALS AND METHODS: ASA presence was evaluated by the MAR test. Their prevalence, impact on semen quality and several aspects of male reproductive function were assessed, retrospectively, in a cohort of 2727 men, divided in 4 groups according to the percentage of ASA (without ASA, 10%-49% ASA, ≥50% ASA, and 10%-100% ASA). RESULTS: The prevalence of ASA was 1.4%. When compared to the group without ASA, patients with ≥50% ASA, presented a higher percentage of semen agglutinates and leukocytes, and higher testicular volume. Subjects with 10%-49% and 10%-100% ASA, had lower levels of FSH and fructose, and higher levels of estradiol, α-glucosidase, and higher testicular volume. Furthermore, the 10%-49% ASA group presented a higher agglutination and percentage of round cells and leukocytes, when compared with the group without ASA. Similar results were observed in the 10%-100% ASA group. Interestingly, smoking habits were more frequent in the ≥50% ASA group, compared with the 10%-49% ASA group, with the chance of having ASA at a pathological level being 2-fold higher. DISCUSSION AND CONCLUSION: In this study, data on ASA prevalence, impact in semen quality and on several aspects of male reproductive health were analyzed in one of the biggest and thoroughly characterized cohorts so far. Agglutination and impaired motility remain the most revealing parameters of ASA presence and should be considered in the clinical ASA management. Interestingly, we are the first to report an association between smoking and pathological ASA levels. The reported higher testicular volume and lower FSH in ASA patients may be of clinical relevance, suggesting that ASA might also have a role in the impaired fertility, highlighting the importance of ASA testing especially in men with lacking etiologic factors.