Elevated palmitic acid (PA) levels are associated with the development of inflammation, insulin resistance (IR) and endothelial dysfunction. Clinopodium chinense (Benth.) O. Kuntze has been shown to lower blood glucose and attenuate high glucose-induced vascular endothelial cells injury. In the present study we investigated the effects of ethyl acetate extract of C. chinense (CCE) on PA-induced inflammation and IR in the vascular endothelium and its molecular mechanism. We found that CCE significantly inhibited PA-induced toll-like receptor 4 (TLR4) expression in human umbilical vein endothelial cells (HUVECs). Consequently, this led to the inhibition of the following downstream adapted proteins myeloid differentiation primary response gene 88, Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon- <mml:math>β</mml:math> and TNF receptor-associated factor 6. Moreover, CCE inhibited the phosphorylation of Ikappa B kinase <mml:math>β</mml:math> , nuclear factor kappa-B (NF- <mml:math>κ</mml:math> B), c-Jun N-terminal kinase, extracellular regulated protein kinases, p38-mitogen-activated protein kinase (MAPK) and subsequently suppressed the release of tumor necrosis factor- <mml:math>α</mml:math> , interleukin-1 <mml:math>β</mml:math> (IL-1 <mml:math>β</mml:math> ) and IL-6. CCE also inhibited IRS-1 serine phosphorylation and ameliorated insulin-mediated tyrosine phosphorylation of IRS-1. Moreover, CCE restored serine/threonine kinase and endothelial nitric oxide synthase (eNOS) activation and thus increased insulin-mediated nitric oxide (NO) production in PA-treated HUVECs. This led to reverse insulin mediated endothelium-dependent relaxation, eNOS phosphorylation and NO production in PA-treated rat thoracic aortas. These results suggest that CCE can significantly inhibit the inflammatory response and alleviate impaired insulin signaling in the vascular endothelium by suppressing TLR4-mediated NF- <mml:math>κ</mml:math> B and MAPK pathways. Therefore, CCE can be considered as a potential therapeutic candidate for endothelial dysfunction associated with IR and diabetes.