Butyrate, a fermentation product of the large bowel microflora, is potentially protective against the development of colorectal cancer. In vitro, butyrate has been shown to induce apoptosis and inhibit proliferation in numerous cancer cell lines, including colorectal cancer. Although these tumor suppressing properties of butyrate are well-documented in experimental systems, the mechanisms underlying the induction of these effects are not fully understood. Understanding these mechanisms in cancer cells, as well as the pathways involved in a cell's ability to overcome them and progress toward malignancy, is vital to determine therapeutic approaches for disease management. We have developed a colorectal cancer cell line (HT29-BR) that is less responsive to the apoptotic effects of butyrate through sustained exposure of HT29 cells to 5 mM butyrate and have used proteomics to investigate the mechanisms involved in the development of butyrate insensitivity. Proteomic analysis identified a number of cellular processes in HT29 and HT29-BR cells influenced by butyrate including remodeling of the actin cytoskeleton, inhibition of protein biosynthesis and dysregulation of the cell stress response. We describe novel roles for butyrate in the induction of its tumor suppressing effects and outline potential cellular pathways involved in the development of butyrate insensitivity in the HT29-BR cell population.