PURPOSE: Recent studies reported therapeutic effects of Smad7 on oral mucositis in mice without compromising radiation therapy-induced cancer cell killing in neighboring oral cancer. This study aims to assess whether a Smad7-based biologic can treat oral mucositis in a clinically relevant setting by establishing an oral mucositis model in dogs and analyzing molecular targets. METHODS AND MATERIALS: We created a truncated human Smad7 protein fused with the cell-penetrating Tat tag (Tat-PYC-Smad7). We used intensity modulated radiation therapy to induce oral mucositis in dogs and applied Tat-PYC-Smad7 to the oral mucosa in dose-finding studies after intensity modulated radiation therapy. Clinical outcomes were evaluated. Molecular targets were analyzed in biopsies and serum samples. RESULTS: Tat-PYC-Smad7 treatment significantly shortened the duration of grade 3 oral mucositis based on double-blinded Veterinary Radiation Therapy Oncology Group scores and histopathology evaluations. Topically applied Tat-PYC-Smad7 primarily penetrated epithelial cells and was undetectable in serum. NanoString nCounter Canine IO Panel identified that, compared to the vehicle samples, top molecular changes in Tat-PYC-Smad7 treated samples include reductions in inflammation and cell death and increases in cell growth and DNA repair. Consistently, immunostaining shows that Tat-PYC-Smad7 reduced DNA damage and neutrophil infiltration with attenuated TGF-β and NFκB signaling. Furthermore, IL-1β and TNF-α were lower in Tat-PYC-Smad7 treated mucosa and serum samples compared to those in vehicle controls. CONCLUSIONS: Topical Tat-PYC-Smad7 application demonstrated therapeutic effects on oral mucositis induced by intensity modulated radiation therapy in dogs. The local effects of Tat-PYC-Smad7 targeted molecules involved in oral mucositis pathogenesis as well as reduced systemic inflammatory cytokines.