TLRs, including TLR4, have been shown to play a crucial role in cardiovascular inflammatory-based diseases. The main goal of this study was to determine the potential of FP7, a synthetic glycolipid active as a TLR4 antagonist, to modulate haematopoietic and non-haematopoietic vascular TLR4 pro-inflammatory signalling. HUVEC, human THP-1 monocytes, THP-1-derived macrophages, mouse RAW-264.7 macrophages and Angiotensin II-infused apolipoprotein E-deficient mice were in vitro and in vivo models, respectively. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 on TLR4 functional activity in response to bacterial LPS ( in vitro) and endogenous ligands of sterile inflammation ( in vitro and in vivo). Following activation of TLR4, in vitro and in vivo data revealed that FP7 inhibited p38 MAPK and p65 NF-kB phosphorylation associated with down-regulation of a number of TLR4-dependent pro-inflammatory proteins. In addition to inhibition of LPS-induced TLR4 signalling, FP7 negatively regulated TLR4 activation in response to ligands of sterile inflammation (hydroperoxide-rich oxidised LDL, in vitro and Angiotensin II infusion, in vivo). These results demonstrate the ability of FP7 to negatively regulate in vitro and in vivo haematopoietic and non-haematopoietic vascular TLR4 signalling both in humans and mice, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of vascular inflammatory diseases.