ObjectiveSecondary metabolites and polyphenolic compounds from medicinal plants have been demonstrated to have multiple biological functions with promising research and development prospects. This study examined the effect of β-stigmasterol (with ergosterol) and xylopic acid isolated from Anchomanes difformis on liver mitochondrial permeability transition pore (mPTP). MethodsThe compounds were isolated by vacuum liquid chromatography. Mitochondrial swelling was assessed as changes in absorbance under succinate-energized conditions. Results1H and 13C NMR spectroscopic elucidation of the isolates affirmed the presence of β-stigmasterol with ergosterol (1:0.3) and xylopic acid. The isolates reversed the increase in lipid peroxidation and inhibited the opening of mitochondrial permeability transition pores caused by calcium and glucose. Pharmacological inhibition of mPTP offers a promising therapeutic target for the treatment of mitochondrial-associated disorders. ConclusionReduction in the activity of calcium ATPase and the expression of Caspase-3 and −9 were observed, suggesting that they could play a role in protecting physicochemical properties of membrane bilayers from free radical-induced severe cellular damage and be useful in the management of diseases where much apoptosis occurs.