Phosphatidylinositol-4,5-bisphosphate (PIP₂) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (Ca_V) channels promotes insulin secretion in pancreatic β-cells. However, whether Ca_V channels are regulated by PIP₂, as is the case for some noninsulin-secreting cells, is unknown. The purpose of this study was to investigate whether Ca_V channels are regulated by PIP₂ depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). Ca_V channel currents were recorded by the patch clamp technique. The Ca_V current amplitude was reduced by activation of the muscarinic receptor 1 (M₁R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced Ca_V inhibition was diminished by a high concentration of Ca²⁺ chelator, whereas ≥ 50% of this inhibition was prevented by diC8-PIP₂ dialysis. Localization of PIP₂ in the plasma membrane was examined by transfecting INS-1 cells with PH-PLC1, which revealed a close temporal association between PIP₂ hydrolysis and Ca_V channel inhibition. Furthermore, the depletion of PIP₂ by a voltage-sensitive phosphatase reduced Ca_V currents in a way similar to that observed following M₁R activation. These results indicate that activation of the M₁R pathway inhibits the Ca_V channel via PIP₂ depletion by a Ca²⁺-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.