Phospholipase Cβ (PLCβ) isozymes represent a family of molecules that link G protein-coupled receptors (GPCRs) to an intracellular signaling network. Here, we investigated the function of PLCβ isozymes in sensory neurons by using mutant mice deficient for specific PLCβ family members. Expression analysis indicated that PLCβ3, one of the four isoforms, is predominantly expressed in a subpopulation of C-fiber nociceptors. A subset of these neurons expressed the histamine H1 receptor. Ca²⁺ imaging studies revealed that PLCβ3 specifically mediates histamine-induced calcium responses through the histamine H1 receptor in cultured sensory neurons. In line with this, we found that PLCβ3^−/− mice showed significant defects in scratching behavior induced by histamine; histamine-trifluoromethyl-toluidine (HTMT), a selective H1 agonist; and compound 48/80, a mast cell activator. These results demonstrate that PLCβ3 is required to mediate "itch" sensation in response to histamine acting on the histamine H1 receptor in C-fiber nociceptive neurons.