Silibinin, a flavonoid isolated from Silybum marianum, has been reported to have cancer chemopreventive and therapeutic effects. Here, we show that treatment with subtoxic doses of silibinin in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, but not in human astrocytes, suggesting that this combined treatment may offer an attractive strategy for safely treating gliomas. Although the proteolytic processing of procaspase-3 by TRAIL was partially blocked in glioma cells, cotreatment with silibinin efficiently recovered TRAIL-induced caspase activation in these cells. Silibinin treatment up-regulated DR5, a death receptor of TRAIL, in a transcription factor CHOP-dependent manner. Furthermore, treatment with silibinin down-regulated the protein levels of the antiapoptotic proteins FLIP(L), FLIP(S), and survivin through proteasome-mediated degradation. Taken together, our results show that the activity of silibinin to modulate multiple components in the death receptor-mediated apoptotic pathway is responsible for its ability to recover TRAIL sensitivity in TRAIL-resistant glioma cells.