Using H-500 rat Leydig cancer cells as a model of humoral hypercalcemia of malignancy (HHM), we previously showed that high Ca²⁺ induces PTH-related peptide (PTHrP) secretion via the calcium-sensing receptor (CaR) and mitogen- and stress-activated kinases, e.g. MAPK kinase 1 (MEK1), p38 MAPK, and stress-activated protein kinase 1/c-Jun N-terminal kinase. Because cellular proliferation is a hallmark of malignancy, we studied the role of the CaR in regulating the proliferation of H-500 cells. Elevated Ca²⁺ has a mitogenic effect on these cells that is mediated by the CaR, because the calcimimetic NPS R-467 also induced proliferation. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 MAPK but not MEK1 abolished the mitogenic effect. Activation of PI3K by elevated Ca²⁺ was documented by phosphorylation of its downstream kinase, protein kinase B. Because protein kinase B activation promotes cell survival, we speculated that elevated Ca ²⁺ might protect H-500 cells against apoptosis. Using terminal uridine deoxynucleotidyl nick end labeling staining, we demonstrated that high Ca²⁺ (7.5 mM) and NPS R-467 indeed protect cells against apoptosis induced by serum withdrawal compared with low Ca²⁺ (0.5 mM). Because the CaR induces PTHrP secretion, it is possible that the mitogenic and antiapoptotic effects of elevated Ca²⁺ could be indirect and mediated via PTHrP. However, blocking the type 1 PTH receptor with PTH (7-34) peptide did not alter either high Ca₂₊-induced proliferation or protection against apoptosis. Taken together, our data show that activation of PI3K and p38 MAPK but not of MEK1/ERK by the CaR promotes proliferation of H-500 cells as well as affords protection against apoptosis. These effects are likely direct without the involvement of PTHrP in an autocrine mode.