Prostate cancer almost exclusively metastasizes to skeletal sites, indicating that the bone provides a favorable microenvironment for its localization and progression. A natural yet understudied factor in bone that could facilitate tumor localization is elevated extracellular calcium ([Ca²⁺]o). The present study found that elevated [Ca²⁺]o (2.5 mmol/L) enhanced proliferation of skeletal metastatic prostate cell lines (PC-3 and C4-2B), but not the nonskeletal metastatic, epithelial-derived prostate cell line LNCaP. The proliferative effect of elevated [Ca²⁺]o was associated with higher expression of the calcium-sensing receptor (CaSR), a heterotrimeric G-protein–coupled receptor that is the predominant cell-surface sensor for [Ca²⁺]o. Knockdown of the CaSR via RNA interference reduced cell proliferation in vitro and metastatic progression in vivo. CaSR signaling in PC-3 cells was evaluated by measuring the elevated [Ca²⁺]o-dependent inhibition of cyclic AMP accumulation, induced by either prostaglandin E₂ or forskolin. Elevated [Ca²⁺]o stabilized expression of cyclin D1, a protein required for cell cycle transition. Furthermore, elevated [Ca²⁺]o triggered activation of the Akt signaling pathway and enhanced PC-3 cell attachment. Both pertussis toxin (a G-protein inhibitor) and LY294002 (an inhibitor of Akt signaling) reduced cell attachment. These data suggest that elevated [Ca²⁺]o following increased bone remodeling could facilitate metastatic localization of prostate cancer via the CaSR and the Akt signaling pathway. Taken together, [Ca²⁺]o is a candidate mediator of prostate cancer bone metastasis. (Cancer Res 2006; 66(18): 9065-73) (Cancer Res 2006; 66(18): 9065-73)