Aim: Berberine is thought to be an immunomodulator, so the present study aimed to investigate the effect of berberine on mortality, lung and intestine injury in endotoxemic mice, and the mechanism of its action. Methods: Mice were challenged with lipopolysaccharide (LPS, 28 mg/kg, ip), and neutral sulfate berberine was administrated intragastrically. Mortality was monitored every 12 h, and histology of the lungs and intestine as well as the plasma tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12 (IL-12), IL-10, and nitric oxide (NO) levels were examined. Results: Pretreatment with 50 mg/kg neutral sulfate berberine once a day for 5 days significantly decreased the mortality rate and attenuated tissue injury of the lungs and small intestine in mice challenged with LPS. LPS stimulated a marked increase in plasma levels of TNF-α, IFN-γ, IL-12, IL-10, and NO. The administration of berberine significantly reduced plasma TNF-α, IFN-γ, and NO levels, but did not suppress plasma IL-12 levels in mice exposed to LPS. Furthermore, pretreatment with neutral sulfate berberine augmented IL-10 secretion stimulated by LPS in mice. Conclusion: Pretreatment with neutral sulfate berberine attenuates tissue injury and improves survival in endotoxemic mice, which may be mediated, at least in part, by the inhibition of pro-inflammatory mediator production and upregulation of IL-10 release. These findings might provide a new strategy for the treatment of endotoxemia.