Objective: Metformin is a first-line pharmacotherapy in the treatment of t ype 2 diabetes, a condition closely associated with non-alcoholic fatty liver disease (NAFLD). Although metfor min promotes weight loss and improves insulin sensitivity, its effect on intrahepatic triglyceride (IHTG) rema ins unclear. We investigated the effect of metformin on IHTG, hepatic de novo lipogenesis (DNL), and fatty acid (FA) oxidation in vivo in humans. Design and methods: Metabolic investigations, using stable-isotope tracers, were p erformed in ten insulin-resistant, overweight/obese human participants with NAFLD who were treatme nt naïve before and after 12 weeks of metformin treatment. The effect of metformin on markers of s.c. adipose ti ssue FA metabolism and function, along with the plasma metabolome, was investigated. Results: Twelve weeks of treatment with metformin resulted in a signific ant reduction in body weight and improved insulin sensitivity, but IHTG content and FA oxidation remained unchanged. Metformin treatment was associated with a significant decrease in VLDL-triglyceride (TG) concentrations and a significant increase in the relative contribution of DNL-derived FAs to VLDL-TG. There were subtle and relatively fe w changes in s.c. adipose tissue FA metabolism and the plasma metabolome with metformin treatment. Conclusions: We demonstrate the mechanisms of action of metformin whereby it improves insulin sensitivity and promotes weight loss, without improvement in IHTG; these observations are partly explained through increased hepatic DNL and a lack of change in FA oxidation. [ABSTRACT FROM AUTHOR]