<p>Oral submucous fibrosis (OSF) is a chronic and progressive fibrosis disease. Although its pathological stages are well characterized in clinical settings, corresponding animal models remain lacking, which has significantly hindered in-depth mechanistic studies and the development of targeted interventions. Herein, we developed new methods in creating OSF models in rats and mice by different injection frequency of bleomycin (BLM) to simulate early and middle stages of fibrosis. The results showed both low-frequency (once a week) and high-frequency (twice a week) of injection can cause significant fibrosis characteristics, including mucosal pallor, limitation of mouth opening, collagen deposition and inflammatory response. Histological and molecular analyses confirm the stage-dependent pathological changes. Furthermore, to validate the pathological relevance of the model, human oral mucosal tissues from OSF patients and healthy individuals were also analyzed. The result exhibits that, compared with low-frequency injections, high-frequency injections of BLM can lead to more serious fibrosis and inflammatory responses in rats and mice, which are corresponding with the early and middle characteristics of human OSF. This work developed stable and repeatable OSF models of rats and mice in different pathological stages, which offer valuable tools for mechanistic studies of OSF and further precise stage-specific therapies.</p>