Abstract: Objective: To explore the mechanism of Guizhou Rosa roxburghii Radix in the treatment of ulcerative colitis in rats based on TLR4/NF-κB signaling pathway. Methods: The ulcerative colitis model was established by gavage with 2,4,6-trinitrobenzenesulfonic acid (TNBS)/ethanol solution, gavage with Rosa roxburghii Radix aqueous decoction high, medium and low dose groups (8, 4 and 2 g/kg) and salazosulfanil group (0.3 g/kg). Observation of the appearance, motor behaviour and blood stool of the rats. Serum and colon of rats were collected, and hematoxylin-eosin (HE) staining was used to observe the pathological changes in the colon of each group of rats. Enzyme-linked immunosorbent assay (ELISA) for the detection of serum interleukin (IL)-1β, IL-6, TNF-α levels in rats. Reverse transcription-polymerase chain reaction (RT-PCR) for the detection of rat colon Myd88, NF-κB p50, NF-κB p65, TLR4 mRNA expression. Protein immunoblotting (Western blot) was performed to detect Myd88, NF-κB p50, NF-κB p65, TLR4 protein expression in rat colon. Results: Rosa roxburghii Radix aqueous decoction significantly improved colonic inflammatory injury in rats with ulcerative colitis, especially in the high dose group of Rosa roxburghii Radix aqueous decoction. Compared with the model group, the Rosa roxburghii Radix aqueous decoction high-dose group showed significant improvement in colon pathological damage. Serum IL-1β, IL-6 and TNF-α levels were significantly decreased in the Rosa roxburghii Radix aqueous decoction high-dose group (P<0.01); colon Myd88, NF-κB p50, TLR4 mRNA expression was significantly decreased (P<0.05); colon Myd88, NF-κB p50, NF-κB p65, TLR4 protein expression was significantly decreased (P<0.01). Conclusion: Rosa roxburghii Radix aqueous decoction can effectively alleviate TNBS-induced ulcerative colitis and ameliorate inflammatory damage. The mechanism of action of Rosa roxburghii Radix aqueous decoction intervention in ulcerative colitis may be related to the inhibition of TLR4/NF-κB signaling pathway.