Background: We have developed a model of alcoholic chronic pancreatitis (necroinflammation+fibrosis) by challenging alcohol-fed rodents with otherwise innocuous doses of lipopolysaccharide (LPS, a bacterial endotoxin). However, the role of the LPS receptor TLR4 in this model is unknown. Aims: i) to compare pancreatic fibrosis in alcohol-fed, LPS challenged wild type (WT) and TLR4 knockout (KO) mice; ii) to determine whether inhibition of TLR4 (via siRNA) decreases activation of pancreatic stellate cells (PSCs - key cells in fibrogenesis) in vitro. Methods : In vivo: WT and KO mice fed Lieber-DeCarli diets ± alcohol for 8 weeks, then challenged with saline or LPS (3 mg/kg IV; 1 injection/week x 3 weeks). Thus, 4 groups (n=4 mice/group) in each set: non-alcohol diet+saline (C), alcohol+saline (A), non-alcohol+LPS (CL), alcohol+LPS (AL). Mice sacrificed 24hr after last injection. Sirius red (collagen) stained pancreatic sections scored for fibrosis. ii) In vitro: Cultured PSCs treated with TLR4 siRNA or scrambled siRNA, then incubated for 48 hours with alcohol (50mM), LPS (1µg/ml), or alcohol+LPS. TLR4 and α smooth muscle actin (αSMA - PSC activation marker) expression assessed by immunoblotting (n=4 PSC preparations). Results: i)In vivo –For WT and KO: fibrosis was negligible in C, A and CL groups. In AL WT mice: fibrosis scores significantly increased over A WT mice (fold change AL over A: 2.603±0.9; p<0.02). In contrast, in AL KO mice: no change in fibrosis over A KO mice (fold change AL over A: 1.01±0.16). ii) In vitro – TLR4 inhibition significantly decreased αSMA expression in LPS and LPS+alcohol treated PSCs (densitometry units scrambled vs TLR4 siRNA 160.9±10.5 vs 80.8±4.4, p<0.01 for LPS; 148.2±11.4 vs 51.4±2.7, p<0.02 for LPS + alcohol). Conclusions: The observed synergistic effects of alcohol + LPS on PSC activation in vivo and in vitro are largely mediated via the LPS receptor TLR4.