AIMS: Electrophilic fatty acid nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long-term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to examine the effects of nitro-fatty acids in the regulation of upstream signalling events in nuclear factor-?B (NF-?B) activation and determine whether low-dose acute administration of nitro-fatty acids reduces vascular inflammation in vivo. METHODS AND RESULTS: Using NF-?B-luciferase transgenic mice, it was determined that pre-emptive treatment with nitro-oleic acid (OA-NO2), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)-induced NF-?B activation both in vivo and in isolated macrophages. Acute intravenous administration of OA-NO2 was equally effective to inhibit leukocyte recruitment to the vascular endothelium assessed by intravital microscopy and significantly reduces aortic expression of adhesion molecules. An acute treatment with OA-NO2 in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS-induced Toll-like receptor 4 (TLR4)-induced cell surface expression in leukocytes and NF-?B activation. In vitro experiments reveal that OA-NO2 suppresses LPS-induced TLR4 signalling, inhibitor of ?B (I?Ba) phosphorylation and ubiquitination, phosphorylation of the I?B kinase (IKK), impairing the recruitment of the TLR4 and TNF receptor associated factor 6 (TRAF6) to the lipid rafts compartments. CONCLUSION: These studies demonstrate that acute administration of nitro-fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro-fatty acids in the disruption of the TLR4 signalling complex in lipid rafts, upstream events of the NF-?B pathway, leading to resolution of pro-inflammatory activation of NF-?B in the vasculature.