Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression, from the early phase of growth arrest to the activation of apoptotic cascades. Previously, we performed 2-D DIGE to identify differentially expressed proteins induced by 24h butyrate treatment of HCT-116 colorectal cancer cells. Herein, we employed quantitative proteomics approaches using iTRAQ, a stable isotope labeling methodology that enables multiplexing of 4 samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cICAT which selectively tags cysteine-containing proteins was also used, and the results complemented that obtained from the iTRAQ strategy. Selected protein targets were validated by real-time PCR and western blotting. A model is proposed to illustrate our findings from this temporal analysis of butyrate-responsive proteome which uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.