Updated project metadata. The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmocotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little of how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we conducted in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. On pathway analysis of the thousands of phosphopeptides identified, we identified “vesicle transport” as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments revealed that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identified several protein binding partners of SEC22B affected by glucagon, some of which were only bound with SEC22B-S137 phosphorylation. In summary, here we demonstrate that phosphorylation of SEC22B is an hepatocellular signaling node mediating the metabolic actions of glucagon, and provide a rich resource for future investigations on the biology of glucagon action.