Hormone sensitive lipase (HSL) is a triacylglycerol hydrolase and cholesterol esterase that is essential for male fertility. The aim of the study was to investigate the distribution of C-60G polymorphism of HSL gene and alleles in fertile and infertile males living in Hamadan, Iran. In addition, lipase activity was determined in these two groups. The HSL genotype was determined by PCR-RFLP and the lipase activity was detected by turbidometery in 164 fertile and 169 infertile males. A significant difference in HSL genotype distribution was observed between groups (χ2 = 8.1, df = 2, p = 0.017). Infertile males showed a higher percentage of CC as well as a lower percentage of CG and GG genotype compared with fertile individuals. The presence of CC to CG + GG genotype conferred a 2.4-fold risk for male infertility (odds ratio = 2.4 (1.3 - 4.5), p = 0.006). In addition, lipase activity was remarkably higher (p < 0.001) in fertile males (94 ± 66 U/L) compared to the infertile subjects (50.6 ± 49 U/L). This suggests that genetic variation of HSL may be a risk factor for male infertility.