Glucose homeostasis is critically dependent on insulin release from pancreatic β-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V_m) and the cytosolic calcium level ([Ca²⁺]_cyt). We propose that TRPM5, a Ca²⁺-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca²⁺-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5^−/− cells. Ca²⁺-imaging and electrophysiological analysis show that glucose-induced oscillations of V_m and [Ca²⁺]_cyt have on average a reduced frequency in Trpm5^−/− islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5^−/− pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5^−/− mice.