Histamine plays a key role in inflammatory responses via increasing chemokine and adhesion molecule productions and augmenting vascular permeability in endothelial cells. Rhodomyrtus tomentosa has been found as a rich source of structurally diverse and biologically active metabolites. In this study, the role of phenolic compound from Rhodomyrtus tomentosa (Aiton) Hassk fruits in down-regulation of histamine-induced EA.hy926 endothelial cell activation was investigated. Herein, myricetin was successfully isolated from R. tomentosa fruits by HPLC and its characterization was identified by mass spectrometer and nuclear magnetic resonance spectroscopy. It was revealed that myricetin was effective in suppression of IL-8 and MCP-1 productions and adhesion molecule generation. Moreover, NF-kappa B activation was inhibited by myricetin via reducing I kappa B-alpha phosphorylation and p50/p65 subunit level. Notably, myricetin potentially attenuated vascular permeability of endothelial cells through decrease in eNOS phosphorylation and intracellular calcium elevation. These results indicate that myricetin from R. tomentosa possesses a promissing pharmaceutical property for the amelioration of endothelial inflammatory responses.