Natriuretic peptide type C (NPPC) activation of the guanylyl cyclase-linked natriuretic peptide receptor (NPR) 2 maintains oocyte meiotic arrest. Luteinizing hormone (LH)-dependent epidermal growth factor (EGF) receptor signaling elevates calcium of cumulus cells to inactivate NPR2, resulting in meiotic resumption. This study investigated the regulatory mechanism of calcium on NPR2 inactivation. In mouse ovarian follicles, LH, through the activation of EGF receptor, significantly elevated calcium levels in cumulus cells, but decreased the binding affinity of NPR2 for NPPC. In cultured cumulus-oocyte complexes, the activation of EGF receptor by EGF mobilized intracellular calcium of cumulus cells to decrease NPR2 affinity and cGMP levels, resulting in meiotic resumption. However, hormone treatments had not changed NPR2 protein levels. In addition, the removal of magnesium ions from the medium decreased the binding affinity of NPR2 for NPPC, resulting in a decrease in cGMP levels and meiotic resumption. It is concluded that magnesium ions are required to maintain functional NPR2, and that LH-dependent EGF receptor signaling mobilizes intracellular calcium of cumulus cells to reduce NPPC-NPR2 interaction that is required for meiotic resumption.