SLC25A47 was initially identified as mitochondrial hepatocellular carcinoma (HCC)-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism.Treatment of hepatocytes with metformin found that metformin can transcriptionally activate the expression of Slc25a47, which is required for AMPKα phosphorylation. Slc25a47-deficient mice had increased hepatic lipid content, triglycerides and cholesterol levels, and we found that Slc25a47-deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in inhibition of lipogenesis. Moreover, using a diethylnitrosamine (DEN)-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mTOR cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD+ was an endogenous substrate for SLC25A47 and the activity of NAD+-dependent SIRT3 declined in Slc25a47-deficient mice, followed by inactivation of AMPKα.Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD+ transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating non-alcoholic fatty liver disease (NAFLD) and HCC.Copyright © 2023 American Association for the Study of Liver Diseases.