The aim of the present study was to evaluate the possible protective effects of thymoquinone (TQ), an antioxidant agent, against imidacloprid (IMI)-induced oxidative stress in male and female mice. In total, 48 Swiss Albino male and female mice were fed a standard rodent diet and divided into 3 equal groups: the animals in the control group (vehicle treated) were given corn oil, the second group were orally administered 15 mg/kg/day IMI alone, and the third group were orally administered 15 mg/kg/day IMI and with TQ at 10 mg/kg/day for 21 days. During the experimental period, there were no significant changes between initial body weights and final body weights of IMI treated male and female mice. IMI produced significant increase in blood, liver, kidney, and heart malondialdehyde (MDA) levels and decrease in blood and liver glutathione (GSH) levels. In addition, IMI treatment decreased erythrocyte, liver, and kidney superoxide dismutase (SOD) activity in male mice and decreased erythrocyte and liver SOD activity in female mice. Erythrocyte catalase (CAT) activities were found to be low in male and female mice. However, treatment with TQ reversed IMI-induced oxidative stress, lipid peroxidation, and activities of antioxidant enzymes. Moreover, TQ exhibited protective action against the IMI-induced histopathological changes in tissues of male and female mice. In conclusion, TQ was found to be effective in protecting mice against IMI-induced oxidative stress by enhancing antioxidant defense mechanisms.