LPS-activated macrophages undergo a metabolic shift from dependence on mitochondria-produced ATP to reliance on aerobic glycolysis, where PKM2 is a critical determinant. Here, we show that PKM2 is a physiological substrate of SIRT5 and that SIRT5-regulated hypersuccinylation inhibits the pyruvate kinase activity of PKM2 by promoting its tetramer-to-dimer transition. Moreover, a succinylation-mimetic PKM2 K311E mutation promotes nuclear accumulation and increases protein kinase activity. Furthermore, we show that SIRT5-dependent succinylation promotes PKM2 entry into nucleus, where a complex of PKM2-HIF1α is formed at the promoter of IL-1β gene in LPS-stimulated macrophages. Activation of PKM2 using TEPP-46 attenuates Sirt5-deficiency-mediated IL-1β upregulation in LPS-stimulated macrophages. Finally, we find that Sirt5-deficient mice are more susceptible to DSS-induced colitis, which is associated with Sirt5 deficiency prompted PKM2 hypersuccinylation and boosted IL-1β production. In conclusion, our findings reveal a mechanism by which SIRT5 suppresses the pro-inflammatory response in macrophages at least in part by regulating PKM2 succinylation, activity, and function. Display omitted •SIRT5 desuccinylates and activates PKM2•Lys311 is a key succinylated site in the regulation of PKM2 activity•Sirt5 blocks IL-1β production in LPS-activated macrophages by regulating PKM2•SIRT5 plays an important role in inhibiting inflammation Activated immune cells reprogram their metabolism in infection or injury response. Wang et al. reveal that SIRT5 desuccinylates and activates PKM2 to block macrophage IL-1β production and to prevent DSS-induced colitis in mice, highlighting the role of SIRT5 and PKM2 in the process of macrophage metabolic reprogramming, sirtuin biology, and inflammation.