Illuminating the mechanisms controlling glucose homeostasis may deepen our understanding of the pathogenesis of T2DM and extend our armamentarium of medications for more specific and tailored treatment of T2DM in future. Employing gain-of-function and loss-of-function approaches, we previously established the role of hepatic Dyrk1b in systemic glucose homeostasis and insulin resitance. To analyze the mechnisms for Dyrk1b in regulation of glucose homeostasis and insulin signaling, TMT-based quantitative proteomics were performed in livers of Dyrk1b overexpression mice and control mice. In this study, we identified 599 differentially expressed proteins (DEPs) in Dyrk1b overexpression mice and verified some of them by western blot. Our findings will advance the understanding of the mechanisms of glycemic control and the pathogenesis of T2DM.