Cholesterol‐rich clusters of SNARE (soluble NSF attachment protein receptor) proteins have been implicated as being important for exocytosis. Here we demonstrate the significance of cholesterol for normal biphasic insulin secretion in mouse β cells by removal of cholesterol from the plasma membrane using methyl‐β‐cyclodextrin (MBCD). Maximal inhibition of insulin secretion in static incubations was achieved using 0.1 mM MBCD. In in situ pancreatic perfusion measurements, both first and second phase insulin secretions were reduced by ~50% (P<0.05). This was accompanied by a reduced number of docked large dense core vesicles (LDCVs) (~40%;P<0.01) and a reduced exocytotic response (>50%; P<0.01). Fur‐ther, subcellular fractionations demonstrated move‐ment of the synaptosomal protein of 25 kDa (SNAP‐25) from the plasma membrane to the cytosol after MBCD treatment. The inhibitory actions of MBCD were coun‐teracted by subsequent addition of cholesterol. We hypothesize that desorption of cholesterol leads to the disturbance of a basic exocytotic mechanism partly due to migration of SNAP‐25, and we conclude that insulin secretion is highly sensitive to changes in plasma mem‐brane cholesterol.—Vikman, J., Jimenez‐Feltstrom, J., Nyman, P., Thelin, J., Eliasson, L. Insulin secretion is highly sensitive to desorption of plasma membrane cholesterol. FASEBJ. 23, 58‐67 (2009)