Obesity has become a global health issue, which can cause metabolic abnormalities systemically leading to increased morbidity of series diseases. At present, researches have presented obesity is a high-risk factor for colitis, and berberine shows positive therapeutic effect on colitis. Thus, we explored the beneficial effects and potential mechanisms of berberine on obesity-exacerbated colitis in this article. High-fat diet (HFD) exacerbated dextran sulfate sodium (DSS) induced colitis mice model was applied, the results showed that HFD promoted DSS-induced weight loss and inflammatory manifestations in intestine. The results of cytokines in serum and mRNA expression of inflammatory indicators in colon showed that HFD increased all their levels evidently, and the outcomes of Western blot analyses presented that HFD downregulated the MFN2 expression, inhibited the phosphorylation of AMPK as well as upregulated the BIP/Grp78 expression, while berberine could significantly reverse all these situations. In vitro, we stimulated Caco-2 cells with palmitic acid (PA) to replicate the lipotoxicity damage in the intestine, and the results presented that intervention therapy of berberine effectively enhanced the MFN2 expression, inhibited the mRNA levels of inflammatory factors, and reversed the PA induced protein level changes of AMPK and BIP/Grp78. In general, we proposed that berberine could regulate MFN2 to alleviate obesity exacerbated colitis. Copyright © 2020. Published by Elsevier Inc.