NOD-like receptors play a crucial role in host defense against intestinal infection. We explored the regulatory effects of berberine on NLRs during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5 d before undergoing cecal ligation and puncture (CLP) to induce polymicrobiol sepsis. The expression of nucleotide-binding oligomerization domain 2 (NOD2), NLR family-pyrin domain containing 3 (NLRP3), the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0 h, 2 h, 6 h, 12 h and 24 h after CLP. Results showed that the Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) level in were significantly lower in berberine treated rats compared to the control animals. The tight junction proteins level, percentage of cell death in intestinal epithelial cells and the mucosal permeability were, on the other hand, significantly elevated in berberine treated rats. The expression of NOD and NLRP3, however, were not significantly affected by berberine treatment. Our results indicate that Pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis but it is likely that the mechanisms of this preventive effect do not involve the NLR pathway.