Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.