Acute exposure to lipopolysaccharide (LPS) can cause hypoglycemia and insulin resistance; the underlying mechanisms, however, are unclear. We set out to determine whether insulin resistance is linked to hypoglycemia through Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor κB (NFκB), a cell signaling pathway that mediates LPS induction of the proinflammatory cytokine tumor necrosis factor alpha (TNFα). LPS induction of hypoglycemia was blocked in TLR4-/- and MyD88-/- mice but not in TNFα-/- mice. Both glucose production and glucose utilization were decreased during hypoglycemia. Hypoglycemia was associated with the activation of NFκB in the liver. LPS inhibition of glucose production was blocked in hepatocytes isolated from TLR4-/- and MyD88-/- mice and hepatoma cells expressing an inhibitor of NFκB (IκB) mutant that interferes with NFκB activation. Thus, LPS-induced hypoglycemia was mediated by the inhibition of glucose production from the liver through the TLR4, MyD88, and NFκB pathway, independent of LPS-induced TNFα. LPS suppression of glucose production was not blocked by pharmacologic inhibition of the insulin signaling intermediate phosphatidylinositol 3-kinase in hepatoma cells. Insulin injection caused a similar reduction of circulating glucose in TLR4-/- and TLR4+/+ mice. These two results suggest that LPS and insulin inhibit glucose production by separate pathways. Recovery from LPS-induced hypoglycemia was linked to glucose intolerance and hyperinsulinemia in TLR4+/+ mice, but not in TLR4-/- mice. Conclusion: Insulin resistance is linked to the inhibition of glucose production by the TLR4, MyD88, and NFκB pathway.