The aim of this work was to investigate the role of mitochondrial dysfunction in the development of oxidative stress and cytotoxicity induced by indomethacin and to evaluate the potential of an apple peel polyphenol extract (APPE) in protecting against these events. Indomethacin induced, time-dependently, mitochondrial and oxidative perturbations which led to cell losses. An inhibition of complex I activity, shown for first time here, which resulted in a concomitant drop in cellular ATP and an increment in mitochondrial superoxide production, was observed after 10 min of exposure. These early cytotoxicity-triggering events were followed by an increase in the intracellular production of superoxide (20 min), an elevation in the activity of xanthine oxidase which led to an increased lipid peroxidation (30 min), and a decline in cell viability which manifested after 40 min. These events were selectively prevented using allopurinol, tempol and APPE (a standardized apple peel polyphenol extract). While the oxidative and cell lytic effects of indomethacin were equally prevented by the three agents, only APPE protected against complex I inhibition and its downstream oxidative consequences. Since tempol (a SOD mimetic) prevented the elevation in xanthine oxidase activity, and allopurinol (a xanthine oxidase inhibitor) totally abolished the increment in lipid peroxidation and loss of cell viability, it appears that a superoxide-dependent increase in xanthine oxidase activity is critical to trigger cytotoxicity. Thus, preventing the early increment in superoxide formation that, as a result of inhibiting complex I, takes place within mitochondria would be key toward protecting the cells against the oxidative and cytolytic effects of indomethacin. The ability of APPE in preventing the inhibition of complex I and the subsequent superoxide-dependent increase in XO activity warrants further studies to evaluate the mechanism involves in the protecting effect of APPE against the indomethacin-associated adverse effects in vivo.