AIM：To explore the potential mechanism of arecoline in promoting oral submucous fibrosis based on key factors of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. METHODS: SD rats were randomly divided into arecoline low-dose group, arecoline medium-dose group, and arecoline high-dose group (5, 10, and 15 mg/mL). The oral buccal mucosa was injected with the corresponding concentration of arecoline (ANE) solution to induce the establishment of oral submucous fibrosis (OSF) models, with 8 rats in each group. Another 8 unmodeled rats were selected as the blank group, and the changes in mouth opening of the rats were detected after 8 weeks of grouping and intervention. HE and Masson staining were used to observe the pathological changes of oral buccal mucosa, measure the length of epithelial staple process and calculate collagen volume fraction. Western blot and qRT-PCR were used to detect the expression of collagen-Ⅰ (COL-I), E-cadherin, fibronectin (FN) and PI3K/Akt/mTOR signaling pathway-related proteins and mRNA in rat oral buccal mucosa.The levels of tumor necrosis factor (TNF) -α, interleukin (IL) -1β and transforming growth factor (TGF) -β1 in rat serum were detected by ELISA. Human immortalized keratinocytes (HaCaT cell line) were cultured in vitro, and the effects of different concentrations of arecoline, PI3K activator, and PI3K inhibitor on the survival rate of HaCaT cells were investigated by CCK-8 method. According to the results of CCK-8, the concentration of arecoline 75 μg/mL, the concentration of PI3K activator 10 μmol/L, and the concentration of PI3K inhibitor 2 μmol/L were selected as the subsequent experimental concentrations. The cells were set as blank group, arecoline group, PI3K activator group, PI3K inhibitor group, and arecoline + PI3K inhibitor group. The mRNA expression levels of COL-I, E-cadherin, FN, PI3K, Akt, and mTOR in each group of cells were detected by qRT-PCR. The levels of TNF-α, IL-1β and TGF-β1 in each group of cells were detected by ELISA. RESULTS: Compared with the blank group, the arecoline low-dose group, the arecoline medium-dose group, and the arecoline high-dose group significantly reduced the mouth opening, significantly shortened the length of the epithelial staple process, significantly increased the collagen volume fraction, inflammatory cell infiltration, and severe pathological damage. The protein expression levels of COL-I, FN, p-PI3K, p-Akt, and p-mTOR were up-regulated, and the protein expression levels of E-cadherin were down-regulated. The mRNA expressions of COL-I, FN, PI3K, Akt, and mTOR were significantly increased. The mRNA expression of E-cadherin was significantly reduced, and the levels of TNF-α, IL-1β, and TGF-β1 were significantly increased (all P<0.05 or P<0.01). Compared with the blank group, the mRNA expression levels of COL-I, FN, PI3K, Akt, and mTOR in the cells of the arecoline group and the PI3K activator group were up-regulated, and the mRNA expression levels of E-cadherin were down-regulated. Compared with the blank group, the mRNA expression levels of COL-I, FN, PI3K, Akt, and mTOR in the cells of the PI3K inhibitor group were down-regulated, and the mRNA expression levels of E-cadherin were up-regulated. Compared with the PI3K inhibitor group, the mRNA expression levels of COL-I, FN, PI3K, Akt, and mTOR in the cells of the arecoline + PI3K inhibitor group were up-regulated, the mRNA expression levels of E-cadherin were down-regulated, and the levels of TNF-α, IL-1β, and TGF-β1 were significantly increased (all P<0.05 or P<0.01). CONCLUSION: Arecoline can significantly promote oral submucous fibrosis, which may play a pro-fibrotic role by upregulating the PI3K/Akt/mTOR signaling pathway.