Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase (mGPD). This study shows that metformin non-competitively inhibits mGPD, altering the hepatocellular redox state and reducing the conversion of lactate and glycerol to glucose, thereby decreasing hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production (EGP) while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide (ASO) knockdown of hepatic mGPD in rats resulted in a phenotype similar to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decrease in plasma glucose concentrations, and inhibition of EGP. These findings were replicated in whole-body mGPD knockout mice. The study also found that metformin inhibits mGPD activity in vitro with an approximate Ki value close to the observed range of plasma metformin levels in patients treated with a normally prescribed dose of one gram of metformin twice a day. Metformin inhibits mGPD non-competitively with a Ki of ~38 µM in rat mGPD and ~55 µM in human mGPD. The study identified mGPD as a primary molecular target by which guanides/biguanides inhibit hepatic gluconeogenesis and provides a novel therapeutic target for type 2 diabetes (T2D). The findings suggest that metformin's blood glucose lowering effects are mediated through alterations in the redox state, rather than through the AMPK pathway. The study also found that metformin inhibits the glycerophosphate shuttle, leading to accumulation of cytosolic NADH, which is unfavorable for conversion of lactate to pyruvate by lactate dehydrogenase (LDH). The study further showed that mGPD ASO treatment abrogated metformin's inhibitory effects on EGP and that mGPD knockout mice had decreased fasting plasma glucose concentrations and decreased rates of EGP compared to wild type littermates. The study also found that metformin inhibits mitochondrial respiration from G-3-P and that the kinetics of metformin inhibition of mGPD was non-competitive. The study concludes that metformin treatment of rats at doses that achieve comparable plasma metformin concentrations observed in metformin-treated T2D patients inhibits mGPD non-competitively and modulates cytosolic and mitochondrial redox state, inducing an effective reduction in EGP. These results identify mGPD as one of the primary molecular targets by which guanides/biguanides inhibit hepatic gluconeogenesis, and provide a novel therapeutic target for T2D.Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase (mGPD). This study shows that metformin non-competitively inhibits mGPD, altering the hepatocellular redox state and reducing the conversion of lactate and glycerol to glucose, thereby decreasing hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production (EGP) while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide (ASO) knockdown of hepatic mGPD in rats resulted in a phenotype similar to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decrease in plasma glucose concentrations, and inhibition of EGP. These findings were replicated in whole-body mGPD knockout mice. The study also found that metformin inhibits mGPD activity in vitro with an approximate Ki value close to the observed range of plasma metformin levels in patients treated with a normally prescribed dose of one gram of metformin twice a day. Metformin inhibits mGPD non-competitively with a Ki of ~38 µM in rat mGPD and ~55 µM in human mGPD. The study identified mGPD as a primary molecular target by which guanides/biguanides inhibit hepatic gluconeogenesis and provides a novel therapeutic target for type 2 diabetes (T2D). The findings suggest that metformin's blood glucose lowering effects are mediated through alterations in the redox state, rather than through the AMPK pathway. The study also found that metformin inhibits the glycerophosphate shuttle, leading to accumulation of cytosolic NADH, which is unfavorable for conversion of lactate to pyruvate by lactate dehydrogenase (LDH). The study further showed that mGPD ASO treatment abrogated metformin's inhibitory effects on EGP and that mGPD knockout mice had decreased fasting plasma glucose concentrations and decreased rates of EGP compared to wild type littermates. The study also found that metformin inhibits mitochondrial respiration from G-3-P and that the kinetics of metformin inhibition of mGPD was non-competitive. The study concludes that metformin treatment of rats at doses that achieve comparable plasma metformin concentrations observed in metformin-treated T2D patients inhibits mGPD non-competitively and modulates cytosolic and mitochondrial redox state, inducing an effective reduction in EGP. These results identify mGPD as one of the primary molecular targets by which guanides/biguanides inhibit hepatic gluconeogenesis, and provide a novel therapeutic target for T2D.