Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcεRI in mast cells triggers the influx of calcium (Ca²⁺) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca²⁺ influx in mast cells. Trpm4^−/− bone marrow–derived mast cells had more Ca²⁺ entry than did TRPM4^+/+ cells after FcεRI stimulation. Consequently, Trpm4^−/− bone marrow–derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4^−/− mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca²⁺ entry in mast cells.