The present study reports beneficial effect of hydroxytyrosol (HT) against arsenic (As)-induced oxidative stress in the rat brain. Rats were orally administered with sodium arsenite dissolved in distilled water (25 ppm, by oral gavage) for 8 weeks or HT (10 mg/kg b. wt.) in combination with As. Results showed increase in protein oxidation and lipid peroxidation, while catalase and superoxide dismutase (SOD) activities as well as GSH content were decreased after As exposure in rat brain. Fourier transform infrared analysis showed significant alteration in peak area values that also validated the oxidative damage to lipids and proteins. In addition, As exposure caused increase in protein expression of caspase-3 and Bax, while Bcl-2 expression was downregulated resulting in translocation of cytochrome c from mitochondria to cytosol. Treatment of HT with As reversed protein oxidation, lipid peroxidation, and increased GSH content as well as catalase and SOD activities. Administration of HT also prevented translocation of cytochrome c from mitochondria and increased mitochondria/cytosol ratio of cytochrome c. Hence, treatment of HT with As improved antioxidant system and efficiently lowered the generation of oxidative stress in rat brain.