Background: Vitamin D is known for its role in calcium metabolism and it has been also shown to be involved in fertility. Previous studies reported an association between the level of vitamin D and semen parameters. There are little studies regrading to this issue in Iranian men. So, we designed this study to assess vitamin D level in Iranian fertile and infertile men. Method: A total of 207 male subjects (112 fertile and 95 infertile) were enrolled in this study. Vitamin D level and semen parameters were compared between the two groups. Results: Mean age, duration of marriage and body mass index (BMI) were not significantly different between the two groups. Frequency of varicocele and previous opioid consumption were significantly higher in the infertile group than the fertile one. Sixty-six (71%) individuals in the infertile group and 61 (55%) in the fertile group had vitamin D levels < 20 (p=0.02). Conclusion: Vitamin D deficiency (< 20) was significantly higher in infertile men.