Insulin is known to increase brain nitric oxide (NO) level but the significance of the increase is not well understood. There is paucity of information on the mechanism by which the increase affects lipid peroxidation and oxidative stress in the brain. The aim of this study was to determine the level of NO and oxidative stress status in the brain of normal non-diabetic mice following sub-acute systemic administration of insulin. Eighteen mice of both sexes, weighing 20.26 ± 1.13 g, were used for the study. They were divided into three groups (n = 6) and treated subcutaneously (s.c.) daily with 0.2 ml deionized water (control), 10 I.U/kg insulin, and 10 I.U/kg insulin (s.c.) + 50 mg/kg L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) intraperitoneally (i.p.) for seven days. Animals were sacrificed following adequate anaesthesia using ketamine + xylazine 65/4 mg/kg, i.p. (Buitrago et al., 2008). Forebrain tissue was immediately harvested from which homogenates were prepared over ice blocks (Shen et al., 2011). Nitric oxide (Biovision, Catalog #262-200) and malondialdehyde (MDA) (Oxford Biomedicals Research, UK, Product number: FR40) levels, as well as glutathione peroxidase (GPx) (Biovision, Catalog #K762-100) activity were determined using commercially available kits according to manufacturers’ instructions. Values were presented as mean ± S.E.M, and compared using ANOVA. Nitric oxide values (nmol/μL) were higher in the insulin group (1.96 ± 0.1) (P < 0.05) but not in the insulin+L-NAME (P > 0.05) group (1.66 ± 0.1) when compared with the control (1.52 ± 0.1). Values of MDA (μΜ) were higher (P < 0.05) and the same (P > 0.05) in the insulin (4.06 ± 0.1) and insulin+L-NAME (3.12 ± 0.1) groups, respectively, compared to those of controls (3.06 ± 0.1). The activity of GPx (nM/min/mg protein) in the insulin group (6.44 ± 0.1) was lower (P < 0.05), but that of the insulin+L-NAME (6.84 ± 0.1) was the same (P > 0.05) when compared with the value obtained in the control group (6.92 ± 0.1). Insulin increased the levels of NO (Kamal et al., 2012) and oxidative stress (Monnier et al., 2011) as indicated by increased MDA, and decreased GPx activity (Agrawal et al., 2009) in the administered mice. This effect was reversed by L-NAME (a non-specific NO inhibitor). These data suggest that insulin increased lipid peroxidation and oxidative stress in the brain through elevation of NO (a free radical) level.