The short chain fatty acids (SCFAs) are inhibitors of histone deacetylases (HDACi); they are produced naturally in the colon by fermentation. They affect cellular processes at a molecular and transcriptional level, the mechanisms of which may involve large numbers of proteins and integrated pathways. Butyrate is the most biologically potent of the SCFAs in colon epithelial cells, inhibiting human colon carcinoma cell proliferation and inducing apoptosis in vitro. In order to investigate the hypothesis that propionate and valerate possess unique and independent actions from butyrate, we combined proteomic and cellomic approaches for large-scale comparative analysis. Proteomic evaluation was undertaken using an iTRAQ tandem mass-spectrometry workflow and high-throughput High-content Analysis microscopy (HCA) was applied to generate cellomic information on the cell cycle and the cytoskeletal structure. Our results show that these SCFAs possess specific effects. Butyrate was shown to have more pronounced effects on the keratins and intermediate filaments (IFs); while valerate altered the β-tubulin isotypes' expression and the microtubules (MTs); propionate was involved in both mechanisms, displaying intermediate effects. These data suggest distinct physiological roles for SCFAs in colon epithelial function, offering new possibilities for cancer therapeutics.