Abstract: Objective: To explore the role of sphingosine kinase 1 (SphK1) in the pathogenesis of arecoline-related oral submucosal fibrosis and its related mechanisms. Methods: An in vitro model of human oral mucosal fibroblasts (BMF) stimulated by arecoline was established,and treated with SphK1 specific inhibitor PF-543,SphK1 overexpression plasmid (SphK1) and Smad2/3 knockdown (shSmad2/3).The expression of epithelial-mesenchymal transition (EMT) markers in cells was detected by Western blotting and immunofluorescence staining,and the migration and invasion abilities of cells were examined by wound healing assay and Transwell invasion assay.The potential pathway of sphK1 regulation in BMF was analyzed by Gene Set Enrichment Analysis. Results: Arecoline increased the expression of SphK1 in BMF in a time- and concentration-dependent manner (P<0.05).Compared with the control group,the number of migrated and invaded cells in the arecoline group was increased (P<0.05),and the expression of epithelial marker E-cadherin was down-regulated while the expression of mesenchymal marker Vimentin was up-regulated in BMF(P<0.05).The number of migrated and invaded cells in the PF-543 group was lower than that in the arecoline group (P<0.05),and the epithelial marker E-cadherin were increased while the expression of mesenchymal marker Vimentin was decreased in BMF (P<0.05).Gene Set Enrichment Analysis showed that transforming growth factor-β (TGF-β) signaling was the most significantly activated pathway upon SphK1 overexpression.Smad2/3 knockdown effectively blocked SphK1-induced cell migration and invasion (P<0.05),and reversed the expression of downstream EMT markers in SphK1-overexpressing cells (P<0.05). Conclusions: SphK1 is involved in the malignant transformation of oral submucosal fibrosis through TGF-β-dependent EMT.