The peptide hormone adropin regulates energy metabolism in skeletal muscle and plays important roles in the regulation of metabolic homeostasis. Besides muscle, the liver has an essential role in regulating glucose homeostasis. Previous studies have reported that treatment of diet-induced obese (DIO) male mice with adropin34-76 (the putative secreted domain) reduces fasting blood glucose independently of body weight changes, suggesting that adropin suppresses glucose production in the liver. Here, we explored the molecular mechanisms underlying adropin's effects on hepatic glucose metabolism in DIO mice. Male DIO B6 mice maintained on a high-fat diet received five intraperitoneal injections of adropin34-76 (450 nmol/kg/injection) over a 48-h period. We found that adropin34-76 enhances major intracellular signaling activities in the liver that are involved in insulin-mediated regulation of glucose homeostasis. Moreover, treatment with adropin34-76 alleviated endoplasmic reticulum stress responses and reduced activity of c-Jun N-terminal kinase in the liver, explaining the enhanced activities of hepatic insulin signaling pathways observed with adropin34-76 treatment. Furthermore, adropin34-76 suppressed cAMP activated protein kinase A (PKA) activities, resulting in reduced phosphorylation of inositol trisphosphate receptor, which mediates endoplasmic reticulum calcium efflux, and of cAMPresponsive element-binding protein, a key transcription factor in hepatic regulation of glucose metabolism. Adropin34-76 directly affected liver metabolism, decreasing glucose production and reducing PKA-mediated phosphorylation in primary mouse hepatocytes in vitro. Our findings indicate that major hepatic signaling pathways contribute to the improved glycemic control achieved with adropin34-76 treatment in situations of obesity.