Colonic butyrate may maintain mucosal differentiation and oppose carcinogenesis. We characterized butyrate effects on differentiation, proliferation, and matrix interactions in Caco-2 and SW620 human colonic cells. Differentiation was assessed by brush border enzyme activity and doubling time by serial cell counts. Motility across matrix proteins was quantitated by monolayer expansion and correlated with adhesiveness to matrix. Integrin subunit surface pools were measured by immunoprecipitation. Butyrate-stimulated differentiation inhibited proliferation and was significantly more potent than acetate in this regard. Butyrate also inhibited motility across collagen I, collagen IV, and laminin, as well as decreasing adhesiveness to these matrices and beta 1, alpha 1, and alpha 2 integrin subunit surface expression. Butyrate acts in cultured cells at clinically relevant concentrations to oppose classical malignant behavior, inhibiting proliferation and motility while promoting differentiation. Since butyrate is derived from fermentation of dietary fiber, such mechanisms may contribute to the apparent protective action of fiber against colon carcinogenesis.