Ion channel is the regulatory mechanism for electrical activity in pancreatic islet cells through stimulus-secretion coupling. Changes in membrane potential are regulated by the glucose concentration-dependent ion channel activities. The alteration of glucose concentration is linked to the open probability of ATP-sensitive K+ channels by insulin secretion. At the meantime, the change of glucose concentration can cause the reorganization of the membrane as well as the cytoskeleton, resulting in the change of cellular stiffness. By using an integrated AFM and cell manipulation system, we were able to measure the cell stiffness and structural change simultaneously upon the glucose stimulation. The cell stiffness increases substantially in a dosage-dependent manner after stimulation by real time AFM nanoindentation measurement. Structurally, the cell height decrease dynamically with the glucose concentration increase. Therefore we have a unique Bio Marker to characterize the ion channel activity using different modalities. This result indicates that the open and close of ion channel would lead to the change of membrane structure and thus the cell body exhibits a different cellular stiffness. The study will enhance our understanding of pancreatic islet cell stimulus coupling and insulin secretion.