Subsequently, <i>in vitro</i> experiments using human primary oral mucosal fibroblasts (hOMFs) were conducted to validate the molecular mechanisms.<h4>Results</h4><i>In vivo</i> experiments demonstrated that chronic topical application of arecoline significantly reduced oral opening distance and induced histopathological features of oral submucous fibrosis (OSF), including epithelial atrophy, collagen deposition, and elevated TGF-β expression. Here, TGFB1 is linked to oral submucous fibrosis.