► 7k-Stigmasterol did not cause deleterious effects in Caco-2 cells. ► The sub-cellular target of 7k-cholesterol toxicity seems to be the mitochondria. ► 7k-Stigmasterol demonstrated a capacity to reduce toxic effects of 7k-cholesterol. The biological implications of cholesterol oxidation products have been investigated, though research on plant sterol oxidation products is scarce and in some cases contradictory. The cytotoxicity of 7keto(k)-stigmasterol versus 7keto(k)-cholesterol at different concentrations (0–120μM) and incubation times (4–24h), in intestinal epithelial cells (Caco-2 cells) was evaluated. The 3-4,5-dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide and neutral red uptake tests, mitochondrial membrane potential (ΔΨm), and relative DNA and RNA contents in the cell cycle phases were determined. Possible interaction effects between 7k-derivatives or non-oxidized stigmasterol were monitored. Endo/lysosomal activity was not impaired by either oxide. 7k-cholesterol showed a deleterious effect upon the mitochondrial compartment after 24h of exposure (120μM), as well as upon ΔΨm when incubated at all concentrations (12/24h). Only cells incubated with 7k-cholesterol (120μM) exhibited a decrease in RNA proportion in the G1 population. The presence of 7k-stigmasterol or stigmasterol with 7k-cholesterol reduced the deleterious metabolic effects upon mitochondrial functionality and integrity and the distribution of RNA contents in G1 and G2 phases. A decrease in the G1 phase proportion was detected in cells exposed to mixtures, without alterations in RNA content. The results obtained indicate the absence of 7k-stigmasterol cytotoxicity in Caco-2 cells and its capacity to reduce 7k-cholesterol toxicity.