Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128-135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.