Endothelial dysfunction is implicated in a variety of cardiovascular diseases although the detailed mechanisms are not yet completely understood. A relationship has been suggested to exist between inflammation and endothelial dysfunction. TNF-α serves as one of the most important pro-inflammatory cytokines. The main objectives of the present study were to explore the effect of PKC-ζ on TNF-α-impaired endothelial function as well as the underlying mechanisms. Acetylcholine-induced endothelium-dependent vasodilation of mouse thoracic aorta stimulated by TNF-α was initially determined. PKC-ζ deficient mice and the specific inhibitor of NADPH oxidase were respectively applied to elucidate their roles in TNF-α-induced endothelial dysfunction. In vitro superoxide generation in HAECs was detected by DHE staining after administration of TNF-α. Meanwhile, the regulatory p47(phox) subunit of NADPH oxidase was evaluated by Western blotting and RT-PCR. The results showed that TNF-α conspicuously impaired endothelium-dependent vasodilation and the impairment was attenuated by either depleting PKC-ζ or inhibiting NADPH oxidase. In vitro TNF-α increased superoxide production and p47(phox) expression in HAECs, and such increases could be ameliorated by the specific PKC-ζ inhibitor. Our findings suggest that superoxide over-production triggered by PKC-ζ-dependent NADPH oxidase activation contributes to TNF-α-induced endothelial dysfunction.