BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAID), which are commonly used to manage pain and inflammation, often cause gastrointestinal injuries, including small intestinal damage. Berberine (BBR) is a traditional Chinese medicine that protects against these injuries. However, the mechanism of action is not fully understood. AIMS: This study aimed to evaluate the protective effects of BBR against NSAID-induced intestinal injury and elucidate the underlying molecular mechanisms. METHODS: We evaluated the effects of BBR on NSAID-induced intestinal injury using a combination of mouse models and human gut organoids. Mice were treated with indomethacin with or without BBR to induce small intestinal injury. Human gut organoids were exposed to NSAID, with or without BBR, to assess their direct epithelial effects. Histological analyses, cytokine measurements, and Western blotting were performed to evaluate intestinal damage, tight junction integrity, and inflammasome-associated activation. RESULTS: In NSAID-treated mice, BBR markedly reduced ulcers and adhesions and preserved ileal Claudin-1, Occludin, and Zonula Occludens-1 (ZO-1) levels. BBR inhibited both NOD-like receptor family pyrin domain-containing 6 and NOD-like receptor family caspase recruitment domain-containing protein 4 inflammasome activation, reducing Caspase-1 maturation and downstream interleukin-1β and tumor necrosis factor-α release. In human gut organoids, BBR demonstrated comparable protective effects by directly mitigating NSAID-induced epithelial barrier disruption caused by Claudin-1 and Occludin downregulation, although it did not restore ZO-1 expression. CONCLUSIONS: BBR effectively prevented NSAID-induced small intestinal injury by maintaining tight junction integrity and inhibiting inflammasome-associated activation, indicating its potential as a therapeutic agent against such damage.