In the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, treatment with TRAIL in combination with subtoxic doses of rottlerin induced rapid apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in these cells, treatment with rottlerin efficiently recovered TRAIL-induced activation of caspases. Treatment with rottlerin significantly decreased Cdc2 activity through the downregulation of cyclin A, cyclin B, and Cdc2 proteins, whereas the sensitizing effect of rottlerin on TRAIL-induced apoptosis was independent of PKCdelta activity. Furthermore, treatment with rottlerin downregulated the protein levels of survivin and X-chromosome-linked IAP (XIAP), two major caspase inhibitors. Forced expression of Cdc2 together with cyclin B attenuated rottlerin-potentiated TRAIL-induced apoptosis by over-riding the rottlerin-mediated downregulation of survivin and XIAP protein levels. Taken together, inhibition of Cdc2 activity and the subsequent downregulation of survivin and XIAP by subtoxic doses of rottlerin contribute to amplification of caspase cascades, thereby overcoming resistance of glioma cells to TRAIL-mediated apoptosis. Since rottlerin can sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells but not human astrocytes to TRAIL-induced apoptosis, this combined treatment may offer an attractive strategy for safely treating resistant gliomas.