Inflammation provokes significant abnormalities in host metabolism that result from the systemic release of cytokines. An early response of the host is hyperglycemia and resistance to the action of insulin, which progresses over time to increased glucose uptake in peripheral tissue. Although the cytokine TNF-α has been shown to exert certain catabolic effects, recent studies suggest that the metabolic actions of TNF-α occur by the downstream regulation of additional mediators, such as macrophage migration inhibitory factor (MIF). We investigated the glycemic responses of endotoxemic mice genetically deficient in MIF (MIF−/−). In contrast to wild-type mice, MIF−/− mice exhibit normal blood glucose and lactate responses following the administration of endotoxin, or TNF-α. MIF−/− mice also show markedly increased glucose uptake into white adipose tissue in vivo in the endotoxemic state. Treatment of adipocytes with MIF, or anti-MIF mAb, modulates insulin-mediated glucose transport and insulin receptor signal transduction; these effects include the phosphorylation of insulin receptor substrate-1, its association with the p85 regulatory subunit of PI3K, and the downstream phosphorylation of Akt. Genetic MIF deficiency also promotes adipogenesis, which is in accord with a downstream role for MIF in the action of TNF-α. These studies support an important role for MIF in host glucose metabolism during sepsis.