Purpose: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models.Experimental Design: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides.Results: Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence-and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASOtreated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. Conclusions:These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients.