Procto-colectomy with an ileal pouch anal anastomosis is performed in Ulcerative Colitis patients as a potential curative surgical option. However, in many patients a non-specific inflammation of the ileal reservoir can occur, named pouchitis. Some patients further develop a chronic antibiotic-resistant disease. Rifaximin, an oral, broad-spectrum antibiotic has been shown to have efficacy for some patients. In the present study CB0125, a novel Rifamycin SV in situ gelling formulation, was developed as a potential pouchitis therapy. This mixture undergoes sol to gel transition under physiological pH and temperature upon administration to the target organ by enema. The in vivo efficacy of the in situ gel was performed using dextran sodium sulphate-induced colitis model in C57/Bl6 mice. The clinical parameters such as body weight changes, rectal bleeding and stool consistency were compared to mesalamine (positive control). In addition, a histopathological investigation was conducted to assess severity of mucosal damage and inflammation infiltrate. CB0125 was well tolerated and there was a significant reduction in disease activity, improved colon weight to length ratio, and improved histology score for the CB-01-25 in situ gel group compared to placebo gel. In addition, CB0125 was superior to mesalamine and to rifamycin SV dissolved in water. We propose that the rifamycin SV in situ gel may provide a longer duration of exposure of this dual acting antibiotic / anti-inflammatory drug at the site of the damaged intestinal mucosa resulting in a superior combined effect, relative to rifamycin SV dissolved in water, for the treatment of pouchitis.