Simple SummaryThe tumor epithelial–mesenchymal transition (EMT) is a critical event in tumor pathogenesis and progression. While MBD3’s significant role in pancreatic cancer EMT has been established, its precise role in colon cancer remains unclear and requires further investigation. Pan-cancer analysis has revealed differential expression of MBD3 in various tumors, significantly associated with tumor occurrence, growth, and progression. Furthermore, analysis of single-cell sequencing and clinical data for colon cancer has revealed a negative correlation between MBD3 expression and clinical indicators such as survival prognosis. Functional enrichment analysis has confirmed the association between MBD3 and EMT in colon cancer. These findings demonstrate MBD3’s potential as a prognostic marker and therapeutic target for colon cancer.AbstractThe tumor EMT is a crucial event in tumor pathogenesis and progression. Previous research has established MBD3’s significant role in pancreatic cancer EMT. However, MBD3’s precise role in colon cancer remains unclear and warrants further investigation. Pan-cancer analysis revealed MBD3’s differential expression in various tumors and its significant association with tumor occurrence, growth, and progression. Moreover, analysis of single-cell sequencing and clinical data for colon cancer revealed MBD3 expression’s negative correlation with clinical indicators such as survival prognosis. Functional enrichment analysis confirmed the association between MBD3 and EMT in colon cancer. Pathological examinations, western blotting, and qRT-PCR in vitro and in vivo validated MBD3’s differential expression in colon cancer. Transwell, CCK-8, clone formation, and in vivo tumorigenesis experiments confirmed MBD3’s impact on migration, invasion, and proliferation. Our findings demonstrate MBD3 as a potential prognostic marker and therapeutic target for colon cancer.