Abstract: Paraquat (PQ), an oxidative stress inducing substance, is a herbicide which is very toxic to the animals and humans. A number of antioxidant treatments against PQ-induced oxidative stress have been tried but unfortunately, none of them has been proven to be enormously effective. In this context, attentions are needed to explore the antioxidants from the natural resources. Thus, the antioxidant activity of Withania somnifera (Ws) root extract on paraquat (PQ) induced oxidative stress in mice was investigated. The doses of 100, 250 and 500 mg kg-1 of Ws were given orally on alternate day for 30 days. PQ (3.5 mg kg-1) was injected intraperitoneally on day 15th of start of the study. On day 30th antioxidant activity of Ws was evaluated by measuring lipid peroxides (MDA) and GSH contents as well as by measuring the activities of antioxidant enzymes; superoxide dismutase (SOD) and catalase (CAT) in the blood of mice. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, creatinine and urea levels were also estimated to investigate the protective effects of Ws on liver and kidney functions. Ws remarkably alleviated the over production of MDA and improved GSH level in PQ intoxicated mice blood dose-dependently. In addition, SOD and CAT activities were improved significantly by Ws treatment. Ws had also considerably alleviated the elevated AST and ALT activities as well as the total bilirubin, creatinine and urea levels in a dose dependent manner. Ws protected the mice against paraquat-induced oxidative stress mainly through alleviating the over production of MDA and modulating the lowered GSH level, SOD and CAT activities. Thus, Ws may constitute an alternative sole or adjunct remedy against paraquat-induced oxidative stress.