Purpose: The MDM2 oncogene, amplified or overexpressed in many human cancers, has been suggested to be a novel target for cancer therapy. We have demonstrated a second-generation antisense antihuman-MDM2 oligonucleotide to have antitumor activity when administered alone or in combination with cancer chemotherapeutic agents. In the present study, we investigated the effect of the antisense oligonucleotide on radiation therapy. Experimental Design: The in vitro radiosensitization activity was determined in cell lines of human cancers of prostate (LNCaP and PC3), breast (MCF-7 and MDA-MB-468), pancreas (PANC-1), and glioma (U87-MG and A172) and its in vivo radiosensitization activity in xenograft models of LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1. Results: In cells containing at least one functional p53 allele (LNCaP, U87-MG, and A172), after specific inhibition of MDM2 expression, p53 and p21 levels were elevated. In LNCaP cells, the Bax level was increased, and Bcl-2 and E2F1 levels were decreased. In PC3 cells that are p53 null, after inhibition of MDM2 expression, Bax and p21 levels were elevated, and E2F1 levels were decreased. On the basis of in vitro clonogenic assay, the antisense oligonucleotide, in a sequence-specific manner, significantly increased radiation-induced antiproliferation effects. It also increased radiation-induced inhibitory effects on tumor growth in SCID or nude mice bearing LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1 xenografts. Conclusions: These results suggest that MDM2 has a role in radiation therapy of human cancers, regardless of p53 status, providing a basis for future development of MDM2 inhibitors, such as antisense oligonucleotides, as radiosensitizers.