Saturated free fatty acids (FFA) can activate inflammatory cascades including the toll-like receptor 4 (TLR4) pathway. TLR4 is expressed by hepatocytes and may help link FFA to altered hepatic gluconeogenesis in type 2 diabetes mellitus. This study examined the role of TLR4 in mediating palmitate effects on the expression of phosphoenolpyruvate carboxykinase (PCK1) and the catalytic subunit of glucose-6-phosphatase (G6PC), rate-determining gluconeogenic enzymes. Human hepatocellular carcinoma cells (HepG2 and HuH7) were incubated in media including 2% bovine serum albumin and 250 to 1000 μM palmitate for 24 h. Signaling mediated by TLR4 was blocked by a TLR4 decoy peptide or small interfering RNA knockdown of TLR4. Palmitate induced dose-dependent increases in PCK1 and G6PC mRNA abundance, which were prevented by the TLR4 decoy peptide. Palmitate doubled PCK1 promoter activity, and TLR4 knockdown ablated this response. Lipopolysaccharide and monophosphoryl lipid A also up-regulated G6PC and PCK1 transcript abundance in a TLR4-dependent manner. Addition of oleate attenuated palmitate-induced increases in G6PC and PCK1 mRNA abundance. Palmitate increased nuclear factor κ-light-chain-enhancer of activated B cells reporter gene activity, which was unaffected by TLR4 blockade, but increased mRNA abundance of hepatocyte-specific cyclic AMP response element binding protein, a transcriptional regulator of PCK1, in a TLR4-dependent manner. Finally, TLR4 activation by palmitate increased subsequent cellular uptake of palmitate, and inhibiting ceramide synthesis ablated palmitate effects on PCK1 mRNA abundance and promoter activity. These results suggest that TLR4 signaling could play a critical role in linking elevated saturated FFA to increased transcription of gluconeogenic genes.