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-alpha serves as one of the most important pro-inflammatory cytokines. The main objectives of the present study were to explore the effect of PKC-zeta on TNF-alpha-impaired endothelial function as well as the underlying mechanisms. Acetylcholine-induced endothelium-dependent vasodilation of mouse thoracic aorta stimulated by TNF-alpha was initially determined. PKC-zeta deficient mice and the specific inhibitor of NADPH oxidase were respectively applied to elucidate their roles in TNF-alpha-induced endothelial dysfunction. In vitro superoxide generation in HAECs was detected by DHE staining after administration of TNF-alpha. Meanwhile, the regulatory p47(phox) subunit of NADPH oxidase was evaluated by Western blotting and RT-PCR. The results showed that TNF-alpha conspicuously impaired endothelium-dependent vasodilation and the impairment was attenuated by either depleting PKC-zeta or inhibiting NADPH oxidase. In vitro TNF-alpha increased superoxide production and p47(phox) expression in HAECs, and such increases could be ameliorated by the specific PKC-zeta inhibitor. Our findings suggest that superoxide over-production triggered by PKC-zeta-dependent NADPH oxidase activation contributes to TNF-alpha-induced endothelial dysfunction.