Innate immune signaling via TLR 4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM 3 ganglioside in human serum is composed of a variety of fatty acids, including long‐chain (LCFA ) and very‐long‐chain (VLCFA ). Analysis of circulating levels of human serum GM 3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA ‐GM 3 increase significantly in metabolic disorders, while LCFA ‐GM 3 serum levels decrease. Specific GM 3 species also correlates with disease symptoms. VLCFA ‐GM 3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR 4‐mutant mice. In cultured monocytes, GM 3 by itself has no effect on TLR 4 activation; however, VLCFA ‐GM 3 synergistically and selectively enhances TLR 4 activation by LPS /HMGB 1, while LCFA ‐GM 3 and unsaturated VLCFA ‐GM 3 suppresses TLR 4 activation. GM 3 interacts with the extracellular region of TLR 4/MD 2 complex to modulate dimerization/oligomerization. Ligand‐molecular docking analysis supports that VLCFA ‐GM 3 and LCFA ‐GM 3 act as agonist and antagonist of TLR 4 activity, respectively, by differentially binding to the hydrophobic pocket of MD 2. Our findings suggest that VLCFA ‐GM 3 is a risk factor for TLR 4‐mediated disease progression