Dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B), a member of the CMGC group of kinases, is linked to metabolic syndrome, though the underlying molecular mechanisms remain unclear. In this study, we show that Dyrk1b expression is induced in the liver by fasting and in diabetic mice. Through both in vivo and in vitro experiments, we demonstrate that DYRK1B promotes hepatic gluconeogenesis and glucose intolerance. Liver-specific Dyrk1b conditional knockout mice were protected from diet-induced hyperglycemia. Mechanistically, DYRK1B interacts with and phosphorylates FOXO1, primarily at Thr467/Ser468, which is essential for its nuclear localization. Additionally, DYRK1B inhibits AKT-mediated FOXO1 phosphorylation at Thr24 and Ser256, enhancing its nuclear retention. DYRK1B-mediated phosphorylation increases the expression of gluconeogenic genes and promotes gluconeogenesis. Further, AZ191, a pharmacological inhibitor of DYRK1B, significantly reduced blood glucose levels in diabetic mice. Collectively, these findings provide new insights into the role of DYRK1B in glucose metabolism and identify it as a new therapeutic target for treating diabetes.