Hepatic glucose production from glycogen breakdown and gluconeogenic substrates is an important physiologic adaptation, but uncontrolled glucose release by the liver contributes to hyperglycemia in diabetes. The liver can produce glucose from several substrates, including amino acids, lactate/pyruvate, and glycerol. Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) plays an important role in gluconeogenesis from lactate/pyruvate, but its role in gluconeogenesis from other substrates is less clear. For example, glycerol can be converted to glucose by two pathways; [1] a direct pathway involving the reversal of several steps in glycolysis and [2] a mitochondrial pathway requiring the import of pyruvate via the MPC. While prior work has shown that compared to wild-type (WT) mice, hepatocyte MPC-/- mice exhibit lower blood glucose concentrations after fasting 24 h, concentrations after fasting 18 h were not different between genotypes in the present study. To determine whether gluconeogenesis from glycerol was affected by MPC deficiency, we administered 13C-glycerol to overnight fasted WT and MPC knockout mice and found that loss of the MPC increased the incorporation of 13C into new glucose. Further analysis determined that MPC deletion reduced the incorporation of 13C into glucose derived from the mitochondrial pathway and hepatic TCA cycle metabolites, but actually increased glycerol-mediated gluconeogenesis via the direct pathway. Suppression of glycerol kinase or overexpression of glycerol-3-phosphate phosphatase markedly reduced glycerol-mediated glucose production from both the direct and mitochondrial pathways, but did not reduce glucose concentrations in fasted MPC knockout mice. These findings indicate that glycerol-mediated gluconeogenesis is not impaired in MPC-deficient hepatocytes and suggest that gluconeogenesis from other organs, likely kidney or intestine, compensates for loss of the hepatic MPC. Disclosure N.Yiew: None. G.J.Patti: None. S.C.Burgess: None. B.N.Finck: Advisory Panel; Cirius Therapeutics, Inc., Stock/Shareholder; Cirius Therapeutics, Inc. D.Ferguson: None. K.Cho: None. S.Deja: None. C.Jarasvaraparn: None. X.Fu: None. A.J.Lutkewitte: None. S.Mukherjee: None. J.M.Singer: None. Funding National Institutes of Health (R01DK104735, R01DK117657, T32DK007120, R35ES028365)