It is known that exogenous gangliosides (GL) inhibit acute inflammatory signals in different cells induced by Escherichia coli lipopolysaccharide (LPS). Until now the mechanisms underlying their effect are unknown. We hypothesize that the anti-inflammatory effect of GL is caused by their ability to modify TLR4 translocation into the lipid rafts. To test this hypothesis, we studied the effect of exogenous GL on LPS-induced inflammatory reactions associated with increased nitric oxide and prostaglandin E2 (PGE2) production in epithelial cells isolated from the frog Rana temporia urinary bladder. It was shown that preincubation of cells with GM1 and GD1a in the concentration range from 100 nm to 50 μM reduced the effect of 25 μg/ml LPS E. coli on the increase of NO and PGE2 production. The effect of LPS was also eliminated in the presence of polymyxin B, capable to interact with lipid A in LPS molecule, which makes it inaccessible for binding to TLR4. The subcellular fractionation of epithelial cells in the sucrose density gradient in combination with immunoblotting revealed that LPS stimulates translocation of TLR4 into the lipid rafts in the cytoplasmic membrane. Preincubation of cells with GM1 or GD1a at concentration 20 μM completely eliminated the effect of LPS. A similar effect was revealed with 1 mM methyl-β-cyclodextrin, a classical destructor of the lipid rafts. The results indicate the existence of a previously unknown mechanism of the anti-inflammatory effect of exogenous GL associated with their ability to interfere with LPS-induced translocation of TLR4 into the lipid rafts preventing LPS signal transduction. It is assumed that the observed effect of GL is based on their incorporation into cytoplasmic membrane and modification of the lipid rafts organization.