Sepsis, a leading cause of death in hospitalized patients, is characterized by lethal systemic inflammatory responses. JAK2 is an essential tyrosine kinase modulating immune responses. However, the implications of JAK2 in infectious disorders remain undetermined. Here, we report that JAK2 inhibitors rescue animals from polymicrobial sepsis in a clinically relevant time frame. JAK2 inhibition with AG490 prevents NF-κB activation, modulates macrophage activation, and restrains the production of inflammatory cytokines. The inhibition of JAK2 blunted TNF production in both macrophages and splenocytes in a concentration-dependent manner. JAK2 inhibition specifically prevents LPS-induced STAT3 tyrosine phosphorylation without affecting serine phosphorylation in macrophages. JAK2 inhibitor prevents the activation of the canonical p65RelA/p50NF-κB1 pathway but not the other NF-κB proteins. In vivo, JAK2 inhibition restrains serum TNF levels by modulating TNF production in the lung and the spleen and protects mice from lethal endotoxemia in a concentration-dependent manner. AG490 also inhibits extracellular release of HMGB1 from macrophages and prevents an increase in serum HMGB1 levels during sepsis. JAK2 inhibition started at 24 h after the onset of sepsis rescued the mice from polymicrobial sepsis. Our study is the first experimental evidence that JAK2 inhibitors may provide a pharmacological advantage for the treatment of sepsis in a clinically relevant time frame.