Objective-On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. Approach and Results-Because homozygous Poldip2 deletion is lethal, Poldip2 mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2 aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2 tissue. Isolated aortas from Poldip2 mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2 mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2 mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. Conclusions-Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.