The intestinal barrier dysfunction and the gut microbiota dysbiosis with excessive progress of inflammation contribute to the occurrence and acceleration of ulcerative colitis (UC). Painong Powder, a traditional Chinese medicine prescription, consists of Aurantii Fructus Immaturus, Paeoniae Radix Alba and Platycodonis Radix, which has been found to defend against colitis, but it is unclear whether its role in preventing UC is related to gut microbiota. This study aims to evaluate the effects of Painong-San extract (PNS) on UC and reveals the mechanisms related to gut microbiota. Firstly, a total of 125 chemical compounds, including 42 flavonoids, 29 triterpenoids, 21 monoterpenoids, 11 polyphenols, 6 limonoids, 5 alkaloids, 4 coumarins and 7 other compounds, were identified from PNS using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Then, the results in vivo studies demonstrated that PNS treatment reduced the weight loss and the disease activity index, prevented colon shortening and alleviated colonic tissue damage in dextran sulfate sodium (DSS)-induced colitis mice. The intestinal barrier damage was repaired after PNS administration through promoting the expression of tight junction proteins (claudin-1, occludin and zonula occludens-1). More interestingly, PNS regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria, such as Oscillospiraceae and Helicobacter, while the probiotic gut microbiota like Romboutsia, Lactobacillus, Bifidobacterium and Akkermansia were increased. Furthermore, PNS remarkably ameliorated colonic inflammatory response through inhibiting intestinal TLR4/NF-κB signaling pathway by down-regulating the protein expressions of TLR4, MyD88, p-NF-κB p65 and p-IκBα. Taken together, PNS effectively improved DSS-induced colitis through the modulation of gut microbiota, restoration of intestinal barrier function and attenuation of TLR4/NF-κB signaling cascades, which may provide a new explanation of the mechanisms of PNS against UC. © 2021