Toll-like receptor 4 (TLR4), and its co-receptor, Myeloid Differentiation Factor 2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of pro-inflammatory pathways. Here we tested the hypothesis that TLR4 and its co-receptor MD-2 play a central role in non-alcoholic steatohepatitis (NASH) and liver fibrosis in non-alcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 (knock-out, KO) received methionine-choline-deficient (MCD) or methionine-choline-supplemented (MCS) diet. In mice of control genotypes MCD diet resulted in non-alcoholic steatohepatitis, liver triglycerides accumulation and increased Thiobarbituric Acid Reactive Substances (TBARS), a marker of lipid peroxidation, compared to MCS diet. These features of NASH were significantly attenuated in MD-2-KO and TLR4-KO mice. Serum alanine aminotransferase (ALT), an indicator of liver injury, was increased in MCD-diet-fed genotype controls but was attenuated in MD-2-KO and TLR4-KO mice. Inflammatory activation, indicated by serum TNFalpha and nictoinamide adenine dinucleotide phosphate (NADPH) oxidase complex mRNA expression and activation, was significantly lower in MCD-diet-fed MD-2-KO and TLR4-KO compared to corresponding genotype control mice. Markers of liver fibrosis (collagen by Sirius red and alphaSMA staining, procollagen-I, TGFbeta1,alphaSMA, MMP2 and TIMP1 mRNA) were attenuated in MD2- and TLR4-KO compared to their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.