The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22^phox, p47^phox, and p67^phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47^phox protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47^phox gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca²⁺ ([ROS]_i and [Ca²⁺]_i), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCɛ translocation peptide inhibitor, and PKCɛ gene deletion, but not the conventional PKC inhibitor GÖ6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and in [ROS]_i in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H₂O₂ removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H₂O₂ increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS]_i through the mitochondrial ROS-PKCɛ signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS]_i and [Ca²⁺]_i as well as contraction in PASMCs.