Objective Colonic short chain fatty acids(SCFA) may maintain colonocyte differentiation and oppose carcinogenesis, although its precise mechanisms remain unclear. The purpose of this study is to investigate SCFA regulation of intestinal epithelial phenotype by characterizing the effects of three kinds of SCFA on differentiation,proliferation,and migration of human colonic adenocarcinoma cell line Caco-2 and discuss its clinical value. Methods Differentiation was assessed by brush border enzyme expression, doubling time (proliferation) calculated directly from serial cell counts and logarithmic transformation method. Cell motility(migration) was quantitated by the expansion of a confluent Caco-2 monolayer(after release from a constraining fence) across bacteriologic plastic dishes precoated with saturating concentrations of type Ⅰ collagen. Results All three SCFA studied altered the Caco-2 phenotype. Treatment with 10 mmol/L SCFA significantly prolonged the cell doubling time, promoted brush border enzyme expression(cathepsin C), and inhibit the motility of the Caco-2 cells. Conclusion The SCFA butyrate, propionate, and acetate inhibit the proliferation and motility of a well-differentiated human colonic cancer cell line while promoting the expression of the differentiation marker cathepsin C. The SCFA produced by bacterial fermentation of dietary fiber may exert a protective effect against the development of colon cancer.