Introduction: The levels of the cellular energy sensor AMP-activated protein kinase (AMPK) have been reported to be decreased via unknown mechanisms in the liver of mice deficient in growth differentiation factor 15 (GDF15). This stress response cytokine regulates energy metabolism mainly by reducing food intake through its hindbrain receptor GFRAL. Objective: To examine how GDF15 regulates AMPK. Methods: Wild-type and Gdf15 -/- mice, mouse primary hepatocytes and the human hepatic cell line Huh-7 were used. Results: Gdf15 -/- mice showed glucose intolerance, reduced hepatic phosphorylated AMPK levels, increased levels of phosphorylated mothers against decapentaplegic homolog 3 (SMAD3; a mediator of the fibrotic response), elevated serum levels of transforming growth factor (TGF)-β1, as well as upregulated gluconeogenesis and fibrosis. In line with these observations, recombinant (r)GDF15 promoted AMPK activation and reduced the levels of phosphorylated SMAD3 and the markers of gluconeogenesis and fibrosis in the liver of mice and in mouse primary hepatocytes, suggesting that these effects may be independent of GFRAL. Pharmacological inhibition of SMAD3 phosphorylation in Gdf15 -/- mice prevented glucose intolerance, the deactivation of AMPK and the increase in the levels of proteins involved in gluconeogenesis and fibrosis, suggesting that overactivation of the TGF-β1/SMAD3 pathway is responsible for the metabolic alterations in Gdf15 -/- mice. Conclusions: Overall, these findings indicate that GDF15 activates AMPK and inhibits gluconeogenesis and fibrosis by lowering the activity of the TGF-β1/SMAD3 pathway.