Background and Aims Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with a relapsing–remitting pattern that belongs to autoimmune disease (AID). The JAK inhibitor tofacitinib is a newly (2018.05) FDA approved drug for UC which is a drug previously used to treat another AID, rheumatoid arthritis. A higher dose of tofacitinib has been associated with a safety warning for increased lipid levels as well as serious infections and malignancy. Berberine has proved to be a promising drug candidate for the treatment of UC and shows activity in hyperlipidemia, bacteria; infections and cancer. We hypothesized that a balance between clinical benefit and side effects might be attained by using a combination of tofacitinib and berberine‐‐with reduced doses of both drugs. We first investigated the preclinical activity of reduced doses of tofacitinib plus berberine in experimental colitis in the mouse. Methods Tofacitinib (30 mg/kg/day), berberine (40 mg/kg/day) and reduced doses of tofacitinib (15 mg/kg/day) plus berberine (20 mg/kg/day) were administered orally for 14 and 30 days to C57BL/6 mice with colitis induced by two schedules of three cycles of 2% dextran sulfate sodium (DSS). Disease severity, colon length, colon and spleen swelling and cytokine expression in colon tissue as well as histology of major organs were examined. Results The results showed that compared to the two drugs alone, half reduced doses of tofacitinib plus berberine showed a comparable effect in ameliorating DSS‐induced colitis severity and colon shortening. Co‐treatment showed a greater antagonism of DSS induced colon and spleen swelling when compared to either drug alone. Similar effects were observed in cytokine levels in the colon. In addition, no potential toxicity to major organs was observed with this combination therapy. Conclusions Our studies provide a preclinical rationale for the combination of reduced dosage tofacitinib and berberine as a possible clinical therapeutic strategy for the treatment of UC. Support or Funding Information This work was funded by the Research Foundation of South China University of Technology (x2yxD2182200), the Natural Science Foundation of Guangdong Province (2018A030310388), Guangdong Medical Science Foundation (A2018363), and China Scholarship Council (Grant No. CSC201806155103).