Aim To investigate the influence of different concentrations of dextran sulfate sodium (DSS) and administration approaches on the establishment of mouse model of acute ulcerative colitis(UC). Methods Mice were randomly divided into the normal control group, 3% DSS free drinking group, 4% DSS free drinking group, 30% DSS intragastric administration group and 40% DSS intragastric administration group. The amount and average dosage of intaken DSS by mice in free drinking group were measured and the intragastric administration group were given the same dosage. The survival rate, disease activity index (DAI), the lengths and pathological changes of colon were observed, and the coefficients of variation of each indicator described above were compared among the groups. Results Except for the normal control group, other groups developed experimental UC. Among these four approaches, 3% DSS solution free drinking showed appropriate incidence, higher animal survival rate and operability and lower cost, while there was no significant difference in the coefficients of variation of DAI between 3% DSS solution free drinking group and the others. Conclusion 3% DSS solution free drinking has more advantages in the establishment of a murine model with acute UC than other approaches.