Abstract Background Anti-Müllerian hormone (AMH) is released by testicular Sertoli cells and of great importance during fetal male sexual development, but less is known about the role of circulating AMH during adulthood. In vitro studies have shown that vitamin D may induce AMH transcription, but a controlled trial investigating the possible effect of vitamin D on serum AMH has not been conducted in men. Methods A single-center, double-blinded, randomized placebo-controlled clinical trial (NCT01304927) conducted in Copenhagen, Denmark. A total of 307 infertile men were included and randomly assigned (1:1) to a single dose of 300,000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Difference in serum AMH was a predefined secondary endpoint. Explorative outcomes were associations between serum AMH and gonadal function in infertile men. The primary endpoint was difference in semen quality and has previously been published. Results Infertile men in the lowest AMH tertile had significantly lower sperm concentration (∆_T3-1 16 mill/mL (228%); P < 0.001), sperm count (∆_T3-1 55 million (262%); P < 0.001), motile sperm count (∆_T3-1 28 million (255%); P < 0.001), progressive motile sperm count (∆_T3-1 18 million (300%); P < 0.001), testis size (∆_T3-1 2.7 mL (16%); P < 0.001), serum inhibin B (∆_T3-1 72 pg/mL (59%); P < 0.001), inhibin B/FSH ratio (∆_T3-1 48 (145%); P < 0.001), and higher FSH (∆_T3-1 2.6 (38%); P < 0.001) than the tertile of infertile men with highest serum AMH. Vitamin D supplementation had no effect on serum AMH compared with placebo treatment. Conclusions In infertile men, low serum AMH is associated with severely impaired gonadal function illustrated by poor semen quality and lower testosterone/LH ratio. Serum AMH in infertile men was not influenced by vitamin D supplementation.