Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6^{4,7,10,13,16,19}) enhances the effect of butyrate by increasing mitochondrial lipid-oxidation and mitochondrial Ca²⁺-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in (i) human-colon-tumor (HCT-116 isogenic p53+/+ vs p53-/-) cells and (ii) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca²⁺ accumulation in both isogenic cell lines. Ca²⁺ accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca²⁺ uptake. In addition, Mito-Q, a mitochondria targeted antioxidant also blocked apoptosis induced by DHA and butyrate. In complimentary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with/ or without butyrate after which the mitochondria-to-cytosol Ca²⁺ ratio and crypt-viability were measured. No significant difference (p>0.05) in basal mitochondrial Ca²⁺ levels was observed between fish oil or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, p<0.001) in mitochondrial Ca²⁺ as compared to corn oil plus butyrate treatment. Based on these data, we propose that the combination of DHA and butyrate compared to butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca²⁺-dependent (intrinsic) pathway.