Metformin, a widely used therapeutic for type 2 diabetes (T2D), reduces hepatic gluconeogenesis without increasing insulin secretion or causing weight gain. The mechanism behind this effect remains unclear. This study demonstrates that metformin non-competitively inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), altering the hepatocellular redox state, reducing the conversion of lactate and glycerol to glucose, and 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 results provide a novel therapeutic target for T2D and suggest that metformin's rapid glucose-lowering effects may be due to its ability to alter the redox state.Metformin, a widely used therapeutic for type 2 diabetes (T2D), reduces hepatic gluconeogenesis without increasing insulin secretion or causing weight gain. The mechanism behind this effect remains unclear. This study demonstrates that metformin non-competitively inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), altering the hepatocellular redox state, reducing the conversion of lactate and glycerol to glucose, and 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 results provide a novel therapeutic target for T2D and suggest that metformin's rapid glucose-lowering effects may be due to its ability to alter the redox state.