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Background: Heme oxygenase (HO) is an antioxidant enzyme ubiquitously present in the body. Oxidative stress is one of the main causes of male infertility. Here, we aimed to analyze the correlation between serum heme oxygenase-1 (HO-1) level and sperm count, motility, normal morphology, and sperm DNA fragmentation index (DFI) in infertile male patients. Methods: In this retrospective cross-sectional study, serum HO-1 concentrations, routine semen parameters, sperm morphology, and DFI were analyzed in 178 patients. Subjects were stratified into high-HO-1 (≥278 ng/L; n=77) and control (<278 ng/L; n=101) groups. Intergroup comparisons and Spearman's correlation analyses were performed. Results: The high-HO-1 group demonstrated significantly increased normal sperm morphology (P<0.05) and reduced sperm DFI (P<0.05) versus controls. No significant intergroup differences existed in sperm concentration or progressive motility (P>0.05). Correlation analysis revealed positive associations between HO-1 levels and normal morphology (r=0.190, P<0.05), and negative correlations with DFI (r= -0.195, P<0.05).Conclusions: We first identified significant correlations between serum HO-1 levels and both sperm DFI and normal morphology in infertile men, suggesting HO-1's protective role in spermatogenesis. Serum HO-1 quantification may offer a novel strategy for male fertility assessment.