Non-alcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome that is characterized by steatosis, inflammation, and fibrosis, and may progress to cirrhosis and hepatocellular carcinoma. Toll-like receptor 2 (TLR2) is a cell surface receptor, which is involved in the recognition of multiple glycolipids and lipoproteins; however, its role in NASH pathogenesis has not been elucidated. We previously reported a NASH model which was induced in wild-type (WT) mice by an adlibitum feeding of high-fat diet (HFD) for 24 weeks and intravenous injection of oxidized low-density lipoprotein (ox-LDL) during 3 weeks prior to their sacrifice. In present study, we investigated the effect of TLR2 in this model by using TLR2 knockout (KO) mice. WT and TLR2 KO mice fed HFD were significantly heavier than those fed regular diet, although no difference in body weight was observed between the two mouse groups. Histological examination of TLR2 KO mice with HFD + oxLDL displayed attenuated hepatic inflammation and fibrosis compared to those in WT mice with HFD + oxLDL. Similarly, a significant decrease in the mRNA expression for pro-inflammatory cytokines and pro-fibrogenic cytokines was detected in the livers of these TLR2 KO animals. In addition, the hepatic gene expression of peroxisome proliferator-activated receptor (PPAR)-γ was significantly elevated in TLR2 KO mice with HFD + oxLDL. However, the liver lesions of TLR2 KO mice with HFD + oxLDL were characterized by a higher degree of steatosis than those in WT mice with HFD + oxLDL. Consistent with histological findings, TLR2 KO mice with HFD + oxLDL showed higher levels of lipid biochemical markers in livers and blood in comparison to WT counterpart. Our findings suggest that TLR2 signaling mediates the development of liver inflammation and fibrosis but contributes to negative regulation of hepatic steatosis in NASH.