Glucose stimulates insulin secretion from pancreatic islet β cells by altering ion channel activity and membrane potential in the β cells. TRPV1 channel is expressed in the β cells and capsaicin induces insulin secretion similarly to glucose. This study aims to investigate the biophysical properties of the β cells upon stimulation of membrane channels using an atomic force microscopic (AFM) nanoindentation system.ATCC insulinoma cell line was used. Cell stiffness, a marker of reorganization of cell membrane and cytoskeleton due to ion channel activation, was measured in real time using an integrated AFM nanoindentation system. Cell height that represented structural changes was simultaneously recorded along with cell stiffness.After administration of glucose (16, 20 and 40 mmol/L), the cell stiffness was markedly increased in a dose-dependent manner, whereas cell height was changed in an opposite way. Lower concentrations of capsaicin (1.67 × 10(-9) and 1.67 × 10(-8) mol/L) increased the cell stiffness without altering cell height. In contrast, higher concentrations of capsaicin (1.67 × 10(-6) and 1.67 × 10(-7) mol/L) had no effect on the cell physical properties.A unique bio-nanomechanical signature was identified for characterizing biophysical properties of insulinoma cells upon general or specific activation of membrane channels. This study may deepen our understanding of stimulus-secretion coupling of pancreatic islet cells that leads to insulin secretion.