An alteration of mitochondrial function can result in disruption of redox homeostasis and is associated with abnormal cancer cell growth. Manganese superoxide dismutase (SOD2) and glutathione peroxidase 4 (Gpx4) are two of the most important antioxidant defense enzymes that protect cells against oxidative stress. We had previously shown that n-3 polyunsaturated fatty acids (PUFA) promote colonocyte apoptosis, a marker of colon cancer risk, in part by enhancing phospholipid oxidation. To elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, we fed heterozygous SOD2(Het), Gpx4(Het), and transgenic Gpx4(Tg) mice diets containing either 15% corn oil by weight (CO, enriched in n-6 PUFA) or 3.5% CO + 11.5% fish oil (FO, enriched in n-3 PUFA) for 4 weeks. Our data showed that (i) genetic predeposition to oxidative stress facilitates apoptosis in the mouse colon (Gpx4(Het) > SOD2(Het) > Wt > Gpx4(Tg)), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4(Het) and SOD2(Het) mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4(Tg) mice by elevating apoptosis to a level observed in wild-type (Wt; control) animals. Complimentary experiments examining colonic mitochondrial bioenergetic profiles indicate that FO-fed mice exhibit a significantly (P < 0.05) increased respiration-induced proton leak relative to control CO treatment. This finding was consistent with a loss of membrane potential in response to chronic oxidative stress and supports the contention that n-3 PUFA alter mitochondrial metabolic activity, thereby enhancing apoptosis and reducing colon cancer risk.