BACKGROUND: Cyanidin-3-O-glucoside (Cy3g) is a natural anthocyanin, showing favorable anti-cancer efficacy in colorectal cancer (CRC). However, its specific mechanism in CRC remains largely unexplored. OBJECTIVE: This study aimed to investigate the underlying mechanisms of Cy3g on CRC. METHODS: Cell viability of human CRC cell lines (SW620, HT29, LS174T, and HCT116) and normal colon fibroblast cell line (CCD-18Co) treated with Cy3g was detected by CCK-8. Effects of Cy3g on malignant characteristics of SW620 cells were determined by CCK-8, EdU, colony formation, wound healing, Transwell, and flow cytometry assays. To further elucidate Cy3g's mechanism in CRC, KLF4 expression was detected by RT-qPCR, and expression of the extracellular signal-related kinase (ERK) and p38 was examined by western blotting. The effects and mechanisms of Cy3g on CRC progression were further validated in a xenograft mouse model. RESULTS: Cy3g significantly inhibited the cell viability of human CRC cell lines but rarely affected the cell viability of normal colon fibroblast. Cy3g dose-dependently inhibited proliferation, migration, and invasion and promoted apoptosis of SW620 cells. Moreover, Cy3g upregulated KLF4 expression and inactivated the ERK/p38 pathway in a concentration-dependent manner. KLF4 knockdown reversed the inhibitory effects of Cy3g on the malignant characteristics of SW620 and expression of ERK and p38. Animal experiments further validated that Cy3g inhibited tumor growth without altering body weight, activated KLF4, and suppressed the ERK/p38 pathway in CRC model mice. CONCLUSION: Cy3g inhibits CRC progression by suppressing the KLF4-mediated ERK/p38 pathway, offering new insights into CRC prevention and treatment.