It is recognized that the interaction between cells and their physical microenvironment plays a fundamental role in controlling cell behaviors and even in determining cell fate. Any change in the physical properties of the extracellular matrix (ECM), such as its topography, geometry, and stiffness, controls this interaction. In the current study, we revealed a potent interconnection between the cell-matrix interaction and cell-cell communication that is mediated by interface stiffness, and elucidated this process in stem cells from human apical papilla (hSCAPs) in terms of mechanosensing, mechanotransduction, and gap junction-mediated cell-cell communication. We first fabricated polydimethylsiloxane (PDMS) substrates with the same topography and geometry but different stiffnesses and found that the cell morphology of the hSCAPs actively changed to adapt to the difference in substrate stiffness. We also found that the hSCAPs secreted more fibronectin in response to the stiff substrate. The focal adhesion plaques were changed by altering the expression of focal adhesion kinase (FAK) and paxillin. The FAK and paxillin bound to connexin 43 and, as a result, altered the gap junction formation. By performing a Lucifer yellow transfer assay, we further confirmed that the interface stiffness mediated cell-cell communication in living hSCAPs through changes in gap junction tunnels. The intrinsic mechanism that mediated cell-cell communication by extracellular stiffness show the great influence of the interaction between cells and their external physical microenvironment and stress the importance of microenvironmental mechanics in organ development and diseases. STATEMENT OF SIGNIFICANCE: Biochemical factors could direct cell behaviors such as cell proliferation, migration, differentiation, cell cycling and apoptosis. Likewise, biophysical factors could also determine cell behaviors in all biological processes. In the current study, we revealed a potent interconnection between the cell-matrix interaction and cell-cell communication by elucidating the whole process from cell mechanosensing, mechanotransduction to gap junction-mediated cell-cell communication. This process occurs in a collective of cells but not in that of a single cell. Biophysical properties of ECM induced cell-to-cell communication indicates the importance of microenvironmental mechanics in organ development and diseases. These findings should be of great interest in all biological fields, especially in biomaterials - cell/molecular biology involved in the interactions between the cell and its matrix.