Energy release from cellular storage is mandatory for survival during fasting. This is achieved through lipolysis and liver gluconeogenesis. We show here that in the mouse, gut-derived serotonin (GDS) is upregulated during fasting and that it favors both mechanisms. In adipocytes, GDS signals through the Htr2b receptor to favor lipolysis by increasing phosphorylation and activity of hormone-sensitive lipase. In hepatocytes, GDS signaling through Htr2b promotes gluconeogenesis by enhancing activity of two rate-limiting gluconeogenic enzymes, FBPase and G6Pase. In addition, GDS signaling in hepatocytes prevents glucose uptake in a Glut2-dependent manner, thereby further favoring maintenance of blood glucose levels. As a result, inhibition of GDS synthesis can improve glucose intolerance caused by high-fat diet. Hence, GDS opposes deleterious consequences of food deprivation by favoring lipolysis and liver gluconeogenesis while preventing glucose uptake by hepatocytes. As a result, pharmacological inhibition of its synthesis may contribute to improve type 2 diabetes.