Ulcerative colitis (UC) is a digestive disease with a high incidence and is difficult to be cured due to its complex etiology. It has evidenced that intestinal barrier dysfunction plays a predominant role in UC. 20(S)-protopanaxadiol saponins (PDS) isolated from Panax notoginseng possess anti-inflammatory and antioxidative activities, suggesting its potential of treating UC. Herein, the therapeutic effects of PDS against UC and underlying mechanisms in the aspect of intestinal barrier dysfunction were investigated in vivo and in vitro. The results showed PDS had protective effects against dextran sulfate sodium–induced colitis, including attenuating weight loss, disease activity index score elevation, colon length shortening, and histological lesions. Additionally, PDS reduced the colonic activity of myeloperoxidase and the cytokine levels of TNF-α, IL-6, and IL-1β, decreased MDA production, and elevated colonic activities of SOD and GSH-Px in the colitis mice. The expressions of proteins related to tight junction (TJ), including ZO-1, claudin-5, occludin, caveolin-1 (Cav-1), and Nrf2 were downregulated, whereas that of Keap1 was upregulated after colitis induction. These changes were reversed by PDS. Cav-1 expression was downregulated in lipopolysaccharides (LPS)- and H₂O₂-induced HCT116 cells, and the expressions of ZO-1, claudin-5, and occludin were suppressed in HCT116 cells stimulated by LPS and H₂O₂ combined with Cav-1 small interfering RNA transfection, which were ameliorated by PDS, suggesting PDS targeted Cav-1 against intestinal barrier damage. Collectively, PDS alleviates inflammatory injury and oxidative stress by regulating the Nrf2/Keap1 pathway, contributing to targeting Cav-1 against intestinal epithelial TJ proteins loss. It suggests PDS might be a promising therapeutic natural product for UC treatment.