The purpose of this study was to explore the therapeutic effect of the oral administration of pseudo-ginsenoside RT4 (RT4) on ulcerative colitis (UC), and to determine the rate of absorption and distribution of RT4 in mice with UC. Balb/c mice were induced using dextran sulfate sodium salts (DSS) to establish the UC model, and 10, 20, or 40 mg/kg of RT4 was subsequently administered via gavage. The clinical symptoms, inflammatory response, intestinal barrier, content of total short-chain fatty acids (SCFAs), and gut microbiota were investigated. Caco-2 cells were induced to establish the epithelial barrier damage model using LPS, and an intervention was performed using 4, 8, and 16 µg/mL of RT4. The inflammatory factors, transient electrical resistance (TEER), and tight-junction protein expression were determined. Finally, pharmacokinetic and tissue distribution studies following the intragastric administration of RT4 in UC mice were performed. According to the results in mice, RT4 decreased the disease activity index (DAI) score, restored the colon length, reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), and boosted the levels of immunosuppressive cytokine IL-10, increased the content of SCFAs, improved the colonic histopathology, maintained the ultrastructure of colonic mucosal epithelial cells, and corrected disturbances in the intestinal microbiota. Based on the results in caco-2 cells, RT4 reduced the levels of TNF-α, IL-6, and IL-1β; protected integrity of monolayers; and increased tight-junction protein expression. Additionally, the main pharmacokinetic parameters (Cmax, Tmax, t1/2, Vd, CL, AUC) were obtained, the absolute bioavailability was calculated as 18.90% ± 2.70%, and the main distribution tissues were the small intestine and colon. In conclusion, RT4, with the features of slow elimination and directional distribution, could alleviate UC by inhibiting inflammatory factors, repairing the intestinal mucosal barrier, boosting the dominant intestinal microflora, and modulating the expression of SCFAs.