The Nox family NADPH oxidases are reactive oxygen species (ROS) generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here, we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE^-/-) mice. Wild-type (C57Bl6/J), ApoE^-/- and Nox2^-/y/ApoE^-/- mice were maintained on a high fat (21%) diet from 5 weeks-of-age until they were 12 or 19 weeks-old. Mice were sacrificed and their aortas removed for measurement of Nox2 expression (Western blotting, immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to N-nitro-L-arginine) and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE^-/- mice prior to appearance of lesions, and this was associated with elevated ROS levels. In developing plaques, macrophages were also a prominent source of Nox2. Absence of Nox2 in Nox2^-/y/ApoE^-/- double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 weeks-of-age. However, en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2^-/y/ApoE^-/- versus ApoE^-/- mice and this was associated with a marked decrease in aortic ROS production and increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability and early lesion development in ApoE^-/- mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis.