Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptotic cell death in a variety of tumor cells or transformed cells, yet, it is relatively non-toxic to most normal cells. Consequently, TRAIL was thought to be a promising agent for cancer therapy. However, recent research reports revealed that many tumors are unresponsive to TRAIL treatment. Apoptotic agents were identified that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. It was demonstrated that MEKK3-siRNA sensitized MCF-7 cells to TRAIL cytoxicity. In addition, we investigated the discrepancy of the expression of MEKK3 in breast cancers. It was concluded that elevated MEKK3 expression is found at high frequencies in breast cancer compared to normal breast tissue. Further experiments on the signal machinery showed that MEKK3-siRNA increased the sensitivity of MCF-7 cells to TRAIL by suppressing the transcription activity of NF-κB, and enhancing the caspase-processing to generate executive apoptotic signals. These findings indicate that down-regulation of MEKK3 by siRNA approaches will lead to successful treatment of human breast cancer with TRAIL.