The role of experimental probiotic Prato cheese containing Lactobacillus casei 01 in the prevention of dextran sodium sulphate (DSS)-induced ulcerative colitis in mice was evaluated. For the DSS in vivo model, mice were divided into six groups. Groups 1–3 represented noninflamed groups and groups 4–6 received a DSS (2%) solution. Mice from groups 1 and 4 intragastrically received 500 μL of phosphate-buffered saline (control groups), while mice from groups 2 and 5 intragastrically received 500 μL of conventional Prato cheese (control groups). Finally, mice from group 3 and 6 intragastrically received 500 μL of Prato probiotic cheese containing L. casei 01 (9.47 log cfu mL−1). Groups treated with probiotic Prato cheese exhibited reduced weight loss caused by the consumption of DSS solution. However, the probiotic cheese failed to reduce the inflammation scores in DSS-induced colitis group. Overall, probiotic Prato cheese was not effective to ameliorate the symptoms of DSS-induced colitis.