Baicalin is one of flavonoid extracts from Scutellaria baicalensis, which has several functions including anti-inflammation, anti-bacteria, antitumor and et al. However, the mechanisms of anti-inflammatory of baicalin in ulcerative colitis is not clear. Mice colitis models were established by dextran sodium sulfate, Mice administrated with baicalin (100 mg/kg) and mesalazine (100 mg/kg) twice daily by intragastric injection for 7 days after colitis induced were defined as treated group. Then the mice were sacrificed and the colon samples were collected. Toll-like receptor-2, 4, 9 were detected by immunohistochemistry. Signaling proteins such as TLR4, MyD88, and NF-κB p65 were analyzed by western blotting. Cytokine's mRNA include TNF-α, IL-6 IL-10 and IL-13 were measured by reverse transcription polymerase chain reaction. Modified disease activity index were used to analyse the severity of the disease by assessed of diarrhea, stool (occult) blood and body weight loss of the mice. Compared with control and model groups, modified disease activity index in baicalin and mesalazine treated, mice decreased gradually. Immunohistochemistry analysis showed the expression of TLR4, but not TLR2 and TLR9, in the mucosa of mice colon were decreased. Western blot analysis showed that in colitis model, the expression of NF-κB p65 and TLR4 decreased (P < 0.05), while the expression of MyD88 increased significantly compared to control group, and MyD88 expression can not be repressed by baicalin (P < 0.05). Baicalin and mesalazine treatment suppressed the expression of TNF-α, IL-6 and IL-13 mRNA (P < 0.05), yet up-regulated the expression of IL-10 mRNA (P < 0.05), compared to the DDS and control groups. Baicalin administration by intragastric injection ameliorates the severity of colon inflammation. The possible mechanism of anti-inflammatory response by baicalin may involve in the blocking of the TLR4/NF-κB-p65/IL-6 signaling pathway.