OBJECTIVE To explore the mechanism of action of coptisine derivative Q3 against dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice from the perspective of changes in the inflammatory signaling pathway TLR4/NF-κB and compositions of the intestinal microbiota. METHODS Fifty mice were randomly divided into normal control group, model group, sulfasalazine (SASP) group, Q3-low dose group, Q3-high dose group, with 10 in each group. Mice in the groups except the control group were orally administered with 2.5% DSS solution to induce UC model. The SASP was given 700 mg·kg^-1·d^-1 of sulfasalazine by intragastric administration, and the Q3 low and high dose groups were given 50 and 100 mg·kg^-1·d^-1 of Q3, respectively. The other groups were given an equal amount of distilled water. After 6 days of administration, the mouse colon tissues were taken for DAI score and length measurement. HE staining was used to detect the degree of pathological damage in each group. 16S rRNA high-throughput sequencing was used to detect changes in intestinal flora in the intestinal contents of mice. Immunohistochemistry and Western blot were used to detect the expression of TLR4 and p-p65 in colon tissue. The protein expressions of TLR4, p-p65 and p-IκBα and the nuclear translocation of NF-κB p65 in IEC6 cells and RAW264.7 cells were detected by Western blot and immunofluorescence. RESULTS In the DSS-induced mouse ulcerative colitis model, compared with the model group, in vivo Q3 could significantly improve the weight loss, colon length, and increase in DAI scores of UC mice. HE staining results showed that Q3 significantly improved the intestinal pathological damages such as tract epithelial damage, crypt structure disorder and goblet cell reduction; immunohistochemistry and Western blot results showed that Q3 could significantly reduce the expression of TLR4 and p-p65 in the colon tissue of mice in the model group. The results of 16S rRNA showed that Q3 could increase the biodiversity of intestinal microbiota and regulate the composition of intestinal microbiota after DSS administration. It was also shown that in vitro Q3 could inhibit the nuclear translocation of NF-κB p65 in both IEC6 and RAW264.7 cells by immunofluorescence. CONCLUSION Q3 can improve intestinal inflammation by inhibiting the TLR4/NF-κB pathway and regulating the composition of intestinal flora, exerting anti-UC effects, which is expected to become a candidate compound for the treatment of UC.