This study investigates the correlation between vitamin D levels and male infertility, exploring potential mechanisms of action. A total of 306 male participants with suspected infertility were included, and their vitamin D levels were categorized into low (<20 ng/mL) and high (≥20 ng/mL). The study found that vitamin D levels were not significantly influenced by demographic parameters. However, vitamin D was positively correlated with type A and B sperm motility and negatively correlated with type C and D sperm motility. Testosterone levels were significantly positively correlated with vitamin D, while no significant correlations were found between vitamin D and other hormones or essential minerals. Bioinformatic analysis identified 85 shared target proteins between vitamin D and male infertility, with functional annotations indicating involvement in infection response, immune response, FOXO, and HIF1 signaling pathways. The study highlights the positive association between sufficient vitamin D levels and improved reproductive health, suggesting potential regulatory mechanisms through various biological processes and pathways. Future studies are needed to validate these findings and develop targeted interventions to optimize vitamin D levels for male reproductive health.This study investigates the correlation between vitamin D levels and male infertility, exploring potential mechanisms of action. A total of 306 male participants with suspected infertility were included, and their vitamin D levels were categorized into low (<20 ng/mL) and high (≥20 ng/mL). The study found that vitamin D levels were not significantly influenced by demographic parameters. However, vitamin D was positively correlated with type A and B sperm motility and negatively correlated with type C and D sperm motility. Testosterone levels were significantly positively correlated with vitamin D, while no significant correlations were found between vitamin D and other hormones or essential minerals. Bioinformatic analysis identified 85 shared target proteins between vitamin D and male infertility, with functional annotations indicating involvement in infection response, immune response, FOXO, and HIF1 signaling pathways. The study highlights the positive association between sufficient vitamin D levels and improved reproductive health, suggesting potential regulatory mechanisms through various biological processes and pathways. Future studies are needed to validate these findings and develop targeted interventions to optimize vitamin D levels for male reproductive health.