Wnt signaling has been implicated in certain inflammatory diseases. However, the biological role in the inflammatory regulation remains to be characterized. We investigated the regulation by Wnt signaling in endothelial cells, which are active participants and regulators of inflammation. Wnt5a induces cyclooxygenase-2 expression and enhances inflammatory cytokines rapidly, whereas Wnt3a shows limited effects, suggesting a role for β-catenin–independent Wnt signaling in the inflammatory endothelial activation. Pulse-like treatment of Wnt5a induces cyclooxygenase-2 more efficiently than continuous treatment. Wnt5a and TNF-α regulate subsets of cytokines overlapping, only partially, with each other. Calcium ionophore enhances endothelial inflammation similarly, whereas calcium chelators and protein kinase C inhibitor block Wnt5a-induced activation, suggesting a role for the Wnt/Ca 2+/protein kinase C pathway in endothelial inflammatory regulation. Wnt5a activates RelA nuclear translocation and DNA binding. Activated blood vessels, histiocytes, and synoviocytes express Wnt5a in atherosclerosis and rheumatoid arthritis but not in normal tissue, supporting the role of Wnt5a as an inflammatory mediator in vivo. Our data suggest that endothelial inflammation is regulated by a dual system consisting of β-catenin–independent Wnt signaling and TNF-α–mediated signaling.