Objective:Colorectal cancer (CRC) is a common cancer that cannot be detected at an early stage and is a major challenge in oncology research. Studies have shown that vitamin D3 has some anti-cancer and preventive effects on colorectal cancer, but the exact anti-cancer mechanism is not clear. We applied the relevant research methods of network pharmacology to speculate and validate the possible potential pharmacological mechanisms of vitamin D3 for the prevention of colorectal cancer, and to provide more theoretical support for the clinical anticancer effects of vitamin D3.Methods:The relevant targets for vitamin D3 and CRC were obtained from the database of drug and disease targets, respectively. The target of vitamin D3 and the target of colorectal cancer were taken to intersect to obtain common targets. Then, the PPI network was constructed. In addition, the pathways of drug-disease interactions were predicted by GO and KEGG enrichment analysis. Finally, the obtained results were verified to ensure the reliability of the experiments.Results:51 targets of vitamin D3 for the prevention of colorectal cancer were obtained. The 10 core targets were obtained from the PPI network. The 10 core targets include: ALB, SRC, MMP9, PPARG, HSP90AA1, IGF1, EGFR, MAPK1, MAP2K1 and IGF1R. The core targets were further validated by molecular docking and animal experiments. The results suggest that vitamin D3 plays a key role in the prevention of CRC through core targets, PI3K-Akt pathway, HIF-1 pathway, and FoxO pathway.Conclusion:This study will provide more theoretical support for vitamin D3 to reduce the incidence of CRC and is important to explore more pharmacological effects of vitamin D3.