Prostate cancer remains a leading cause of cancer-related death in men, largely attributable to distant metastases, most frequently to bones. Despite intensive investigations, molecular mechanisms underlying metastasis are not completely understood. Among prostate cancer-derived factors, parathyroid hormone-related peptide (PTHrP), first discovered as an etiologic factor for malignancy-induced hypercalcemia, regulates many cellular functions critical to tumor growth, angiogenesis, and metastasis. In this study, the role of PTHrP in tumor cell survival from detachment-induced apoptosis (i.e. anoikis) was investigated. Reduction of PTHLH (encoding PTHrP) gene expression in human prostate cancer cells (PC-3) increased the percentage of apoptotic cells when cultured in suspension. Conversely, overexpression of PTHrP protected prostate cancer cells (Ace-1 and LNCaP, both typically expressing low or undetectable basal PTHrP) from anoikis. Overexpression of nuclear localization signal (NLS)-defective PTHrP failed to protect cells from anoikis, suggesting that PTHrP-dependent protection from anoikis is an intracrine event. A PCR-based apoptosis-related gene array showed that detachment increased expression of the TNF gene (encoding the proapoptotic protein tumor necrosis factor-α) fourfold greater in PTHrP-knockdown PC-3 cells than in control PC-3 cells. In parallel, TNF gene expression was significantly reduced in PTHrP-overexpressing LNCaP cells, but not in NLS-defective PTHrP overexpressing LNCaP cells, when compared with control LNCaP cells. Subsequently, in a prostate cancer skeletal metastasis mouse model, PTHrP-knockdown PC-3 cells resulted in significantly fewer metastatic lesions compared to control PC-3 cells, suggesting that PTHrP mediated antianoikis events in the bloodstream. In conclusion, nuclear localization of PTHrP confers prostate cancer cell resistance to anoikis, potentially contributing to prostate cancer metastasis.