Background: Animal models and observational studies have suggested a favorable role of vitamin D in male reproduction. However, randomized clinical trials investigating the effect of vitamin D supplementation on male fertility are limited. Therefore, this study aimed to examine the effect of vitamin D supplementation on semen quality, reproductive hormones, and anthropometric measurements in vitamin D deficient males with Asthenozoospermia. Methods: Forty-four males with infertility were randomly assigned to the vitamin D group (DG, supplemented with 9 pearls of vitamin D containing 50000 IU vitamin D3 once a week for 12 weeks) and placebo group (PG, received 9 pearls of placebo once a week for 12 weeks). Semen quality markers (sperm count, morphology, sperm motility, semen volume), total testosterone, sexual hormone binding globulin (SHBG), free androgen index (FAI), and anthropometric measurements (weight, body mass index (BMI), waist circumference (WC), hip circumference (HC), and waist to hip ratio (WHR) were measured at the baseline and the end of the study. Results: Serum 25-OH-D levels were significantly higher in the DG compared with the PG. In a multivariate adjusted model, WC significantly decreased in the DG in comparison to the CG (-0.90 ± 0.67 cm VS 0.49 ± 0.38 cm). A marginally-significant increase was observed after vitamin D supplementation for SHBG compared to the baseline value in DG (11.69 ± 5.79, P = 0.05). Compared to the baseline value, sperm immotile was decreased after vitamin D supplementation in the DG (-12.35 ± 5.13, P = 0.02). However, no statistical significant differences were observed in the semen quality markers (sperm count, morphology, motility, and volume), total testosterone, free androgen index, and other anthropometric values. Conclusions: Vitamin D supplementation did not improve semen quality markers, reproductive hormones, and other anthropometric measurements in vitamin D–deficient infertile men compared to the control group.