Abstract: Objective Nuclear translocation of VGLL3 is central to its transcriptional regulation and chemosensitivity modulation, yet the underlying mechanism remains elusive. This study investigates how extracellular matrix stiffness, TGF-β, and etoposide (ETO) regulate VGLL3 nuclear translocation. Methods Ovarian cancer SKOV3 cells were cultured on polydimethylsiloxane (PDMS) substrates of varying stiffness and treated with TGF-β, ETO, or their combination. focal adhesion kinase (FAK) phosphorylation was measured by Western blotting..VGLL3 subcellular localization and nucleocytoplasmic ratio were detected by immunofluorescence. and a FAK-specific inhibitor was used to verify the role of FAK phosphorylation in VGLL3 nuclear translocation. Results Substrate stiffening, TGF-β, and ETO independently induced FAK activation and VGLL3 nuclear translocation, which were effectively blocked by FAK inhibition. ETO-induced VGLL3 nuclear entry was transient (peaking at approximately 30 min followed by a decline), whereas substrate stiffening and TGF-β induced sustained nuclear accumulation of VGLL3. On soft substrates, TGF-β and ETO synergistically promoted VGLL3 nuclear entry through FAK activation; however, on stiff substrates, the stimulatory effects were markedly attenuated. Conclusions This study reveals a mechano-chemical coupling mechanism by which substrate stiffness, TGF-β, and ETO synergistically drive VGLL3 nuclear translocation via FAK phosphorylation, providing new insights into how tumor mechanical microenvironment remodels gene transcriptional regulation to influence chemoresistance and establishing a theoretical basis for targeting the FAK-VGLL3 axis to reverse drug resistance in cancer.