Radiotherapy combined with chemotherapy is the treatment of choice for glioblastoma and locally advanced lung cancer, but radioresistance of these two types of cancer remains a significant therapeutic hindrance. To identify molecular target(s) for radiosensitization, we screened a small interfering RNA (siRNA) library targeting all protein kinases and E3 ubiquitin ligases in the human genome and identified tumor necrosis factor receptor–associated factor 2 (TRAF2). Silencing of TRAF2 using siRNA caused a significant growth suppression of glioblastoma U251 cells and moderately sensitized these radioresistant cells to radiation. Overexpression of a really interesting new gene (RING)–deleted dominant-negative TRAF2 mutant also conferred radiosensitivity, whereas overexpression of wild-type (WT) TRAF2 significantly protected cells from radiation-induced killing. Likewise, siRNA silencing of TRAF2 in radioresistant lung cancer H1299 cells caused growth suppression and radiosensitization, whereas overexpression of WT TRAF2 enhanced radioresistance in a RING ligase-dependent manner. Moreover, siRNA silencing of TRAF2 in UM-SCC-1 head and neck cancer cells also conferred radiosensitization. Further support for the role of TRAF2 in cancer comes from the observations that TRAF2 is overexpressed in both lung adenocarcinoma tissues and multiple lung cancer cell lines. Importantly, TRAF2 expression was very low in normal bronchial epithelial NL20 cells, and TRAF2 silencing had a minimal effect on NL20 growth and radiation sensitivity. Mechanistically, TRAF2 silencing blocks the activation of the nuclear factor-κB signaling pathway and down-regulates several G₂-M cell cycle control proteins, resulting in enhanced G₂-M arrest, growth suppression, and radiosensitization. Our studies suggest that TRAF2 is an attractive drug target for anticancer therapy and radiosensitization. [Cancer Res 2008;68(18):7570–8]