Objective To investigate whether biochanin A （BCA） has a protective effect against dextran sodium sulfate（DSS）-induced ulcerative colitis（UC） in mice. Methods Thirty C57BL/6N mice were randomly divided into normal control group，DSS model group，sulfasalazine（SASP）-positive drug control group，and low/medium/high-dose（5 mg/kg，10 mg/kg，and 20 mg/kg） BCA groups. The mouse model of UC was induced by administering 2.5% DSS aqueous solution for 7 days. During the experimental period，both the normal control and model groups were given 0.5% carboxymethyl cellulose sodium solution daily by gavage. The positive control group was given 100 mg/kg SASP，while the BCA groups were given BCA suspensions at doses of 5 mg/kg，10 mg/kg，and 20 mg/kg. The administration lasted for 10 days. Body weight changes and fecal status of the mice were recorded every day； the colon was dissected，collected，and measured for its length. The colon was stained with Hematoxylin-Eosin for pathomorphological study. Quantitative polymerase chain reaction was used to determine the levels of tumor necrosis factor-α（TNF-α），interleukin-6（IL-6），and interleukin-10 （IL-10） in the colon. Immunofluorescence was used to determine the expression of tight junction proteins，zonula occluden-1 （ZO-1） and occludin，in the colon. Results It showed that 5 mg/kg and 10 mg/kg BCA significantly alleviated weight loss in mice with UC，while 5 mg/kg，10 mg/kg，and 20 mg/kg BCA reduced the disease activity index scores. Additionally，BCA showed similar effects to SASP in improving the structure and reducing the shortening of the colon in mice with UC. Compared with the model group，all BCA groups had significantly decreased TNF-α（P=0.024、P=0.060、P=0.003） and IL-6（P=0.002、P<0.001、P<0.001）and significantly increased IL-10（P=0.006、P=0.003、P<0.001），with varying degrees of up-regulated expression of tight junction proteins. Conclusion BCA can effectively alleviate DSS-induced symptoms，reduce intestinal damage，and protect the intestinal barrier in mice with UC.