Although previous studies have indicated important roles of palmitate, a saturated fatty acid, in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), it remains unclear how palmitate contributes to inflammation and fibrosis in the liver. Administration of palmitate in high fat diet (HFD)-fed but not basal diet (BD)-fed mice resulted in an increase in serum alanine aminotransferase (ALT) levels. Surprisingly, combined administration of very low dose lipopolysaccharide in palmitate-treated mice led to a marked increase in serum ALT levels despite BD-fed conditions. Administration of palmitate alone in BD-fed mice caused inflammatory cell infiltration and liver fibrosis mediated by the toll-like receptor 4 pathway without ALT elevation. In addition, a significant correlation between serum free fatty acid levels and liver fibrosis stage was observed in patients with NAFLD. These results indicate that palmitate may play crucial roles in the pathogenesis of NAFLD in the presence of gut-derived endotoxin.