The vascular endothelium compensates for oxidant stress by increasing the activity of antioxidant enzymes such as glucose‐6‐phophate dehydrogenase (G6PD). G6PD provides reducing equivalents of NAPDH to maintain glutathione stores and modulates nitric oxide synthase (eNOS) activity. To determine whether deficient G6PD activity perturbs these responses, we treated bovine aortic endothelial cells with dehydroepiandrosterone or an antisense oligodeoxynucleotide to G6PD mRNA to decrease G6PD activity and expression. When exposed to hydrogen peroxide, reactive oxygen species (ROS) accumulation was increased in G6PD‐deficient cells compared with those with normal activity. To determine the source of increased oxidant stress in G6PD‐deficient cells, we used inhibitors of ROS generation, which suggested that eNOS was contributing to ROS production. Treatment with L‐NMMA, an inhibitor of eNOS mediated‐nitric oxide (NO) but not superoxide, production confirmed this observation; in contrast to L‐NAME, L‐NMMA promoted ROS generation in G6PD‐deficient cells. In addition, deficient G6PD activity was associated with a decrease in endothelium‐derived bioavailable NO in response to the agonists A23187 and bradykinin as demonstrated by decreased endothelial cGMP and nitrate/nitrite levels. Enhanced ROS accumulation and decreased NO bioavailability may represent one mechanism by which G6PD deficiency contributes to vascular oxidant stress and endothelial dysfunction.