Abstract Stimulation of dendritic cells (DCs) through toll-like receptors (TLRs) is accompanied by an increased metabolic demand, which provides metabolites and energy required for DC activation. This metabolic requirement is fulfilled by TLR-driven rapid glycolytic burst, and inhibition of glycolysis impairs the survival and effector function of DCs. Previous studies have shown an important role for extracellular glucose uptake via glucose transporter 1 (Glut1) in supporting TLR-driven glycolysis; however, the contributions of intracellular glucose stores to these processes have not been well characterized. While non-immune cells store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen in DCs has not been identified. We propose here that cell-intrinsic glycogen metabolism in DCs supports the early glycolytic reprogramming essential for TLR-driven activation. Our data indicate that glycogen metabolism supports the activation of DCs, particularly before the upregulation of Glut1. Here, we show that DCs express the rate-limiting enzymes for glycogen metabolism and possess intracellular glycogen stores. Inhibition of glycogen breakdown severely impairs the expression of key maturation-associated molecules as well as the ability of DCs to stimulate T cells. Additionally, our metabolomics data indicate that the glycogen metabolism in DCs generates both glycolytic and TCA cycle intermediates and that glycogen-derived carbons preferentially engage in metabolic pathways distinct from free glucose catabolism. Our work reveals a novel metabolic regulatory pathway by which DCs differentially utilize glycogen and glucose metabolism to support their activation.