Objective: Pryuvate dehydrogenase kinase 4 (PDK4) is known to be increased in diabetic conditions as well as in the fasting condition when glucagon action is augmented. However, the possibility that PDK4 deficiency or inhibition might also reduce expression of gluconeogenic enzymes has not been properly evaluated. This study was conducted to study besides its effect on limitation of substrate for gluconeogenesis previously observed in global PDK4 KO mice, liver-specific inhibition of PDK4 is sufficient to modulate glucagon-simulated hepatic gluconeogenesis. Methods: Hepatic glucose production and gluconeogenic enzymes were analyzed in primary hepatocyte transfected with Ad-PDK4 or Ad-Mock as well as sh-GFP and sh-PDK4. The rate of fatty acid oxidation (FAO) was measured by metabolic flux analysis using U-[13C]-labeled palmitate. ATP level and cAMP level was determined by LC-MS/MS and ELISA kit, respectively. Results: PDK4 overexpression increased cAMP level in the hepatocyte and therefore stimulated glucose production. Knockdown of PDK4 decreased cAMP level. Metabolic flux anlaysis showed that PDK4 abundance increases the FAO rate which was normalized in the presence of the CPT-1 inhibitor etomoxir. Gain-of-function and loss-of-function studies revealed that PDK4 abundance correlated with glucose production rate as well as FAO. The PDK4-induced increase in ATP was cancelled by etomoxir, suggesting that increased ATP production in PDK4-overexpressed hepatocyte was driven by increased FAO. Causal relationship between FAO flux and cAMP level was analyzed by detecting changes in p-AMPK and p-PDE4B levels after Cpt-1 inhibition. It restored p-AMPK and p-PDE4B level as a result of decreased ATP level and the corresponding increase in AMP/ATP ratio by etomoxir. Here, cAMP and gluconeogenic enzymes were also normalized. Conclusion: PDK4 stimulates glucose production in the liver by promoting FAO and thereby increasing cellular ATP level. Corresponding decrease of p-AMPK and p-PDE4B is sufficient to accumulate cAMP level in the hepatocyte.