Regulators of G-protein signaling (RGS) proteins are regulators of Ca2＋ signaling that accelerate the GTPase activity of the G-protein Ձ-subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced Ca2＋ oscillations. However, the role of RGS4 in Ca2＋ signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced Ca2＋ signaling in pancreatic acinar cells derived from RGS4−/− mice. RGS4−/− acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of Ca2＋ oscillations. RGS4−/− cells also showed increased expression of sarco/endoplasmic reticulum Ca2＋ ATPase type 2. However, there were no significant alterations, such as Ca2＋ signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from RGS4−/− mice. These results indicate that RGS4 protein regulates Ca2＋ signaling in mouse pancreatic acinar cells.