ETHNOPHARMACOLOGICAL RELEVANCE: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H(2)O(2) and its potential molecular mechanisms need larger studies. AIM OF THE STUDY: We established an IPEC-J2 cell damage model induced by H(2)O(2) to explore the protective mechanism of koumine on intestinal injury. MATERIALS AND METHODS: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H(2)O(2)-induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H(2)O(2)-trigged IPEC-J2 cell damage. RESULTS: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H(2)O(2)-induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1beta, IL-6, IL-8, TNF-alpha and TGF-beta), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H(2)O(2). Moreover, koumine dramatically attenuated H(2)O(2)-triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-KappaB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H(2)O(2)-stimulated IPEC-J2 cell inflammation. CONCLUSIONS: This in vitro experimental study suggests that koumine suppresses the H(2)O(2)-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases. CI - Copyright (c) 2022 Elsevier B.V. All rights reserved.