Ethyl pyruvate (EP) has been shown to be an effective anti-inflammatory agent. Herein, we sought to test the following hypotheses: 1) the pharmacological effects of EP persist after cells have been exposed to the compound in vitro, even if the cultures are washed to minimize the amount of EP that is retained in the media; 2) the pharmacological effects of EP persist in vivo, even after waiting a prolonged period (i.e., 6 h) after the last dose of the compound; and 3) the in vivo pharmacological effects of EP are distinct from those of the closely related compound, sodium pyruvate. Incubation of Caco-2 human enterocyte-like monolayers with cytomix, a mixture of interleukin-1beta, interferon-gamma, and tumor necrosis factor, increased permeability to the fluorescent macromolecule, FITC-labeled Dextran (mol wt 4,000 Da). Co-incubation of the cells with 5 mM EP ameliorated cytomix-induced hyperpermeability and induction of iNOS mRNA expression. EP was associated with similar pharmacological effects when cells were pre-incubated with the compound for 24 h prior and then washed extensively prior to adding the cytokine cocktail. Injecting C57Bl/6 mice with lipopolysaccharide (LPS) resulted in gut barrier dysfunction and hepatocellular injury. Although equivalent doses of both EP and sodium pyruvate ameliorated these phenomena, EP was more efficacious than pyruvate. Pretreatment with EP ameliorated the deleterious effects of LPS, even when the duration between the last dose of EP and the endotoxic challenge was 6 h. We conclude that EP provides durable protection against some of the deleterious effects of LPS or pro-inflammatory cytokines.