Korean mistletoe (Viscum album var. coloratum, KML) offers remarkable therapeutic potential for a variety of diseases. This study aims to evaluate the effects and potential molecular mechanisms of KML ethanol extracts (KMLE), focusing on intestinal barrier function and tight junctions (TJs) in an interleukin (IL)-6-induced Caco-2 cell monolayer model and a dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mouse model. KMLE is non-cytotoxic in Caco-2 cells and demonstrated strong antioxidant activity. KMLE alleviated significant barrier dysfunction and protected tight junction proteins (TJPs) in vitro. Furthermore, KMLE alleviated clinical symptoms and histopathological damage, upregulated TJPs, and suppressed the inflammatory cytokines in vivo. Additionally, six bioactive compounds were identified in KMLE by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, KMLE ameliorated intestinal barrier dysfunction in vitro and in vivo. These findings underscore the potential of KMLE as a therapeutic agent for UC, providing insights into the mechanisms through anti-inflammatory properties and its ability to restore TJ integrity.