Whether hispidulin, a flavone from traditional Chinese medicine, can modulate the anticancer effects of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), the cytokine currently in clinical trials was investigated. In the present study, we found that hispidulin potentiated the TRAIL-induced apoptosis in human ovarian cancer cells and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that hispidulin was highly effective in activation of caspases 8 and caspase 3 and consequent poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, we found that hispidulin downregulated the expression of Mcl-1, Bcl-2, and Bcl-xL. Whereas the downregulation of Bcl-2 and Bcl-xL was less pronounced, the downregulation of Mcl-1 was quite dramatic and was time-dependent. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. Interestingly, we determined that AMPK is activated upon hispidulin treatment, resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease. Therefore, our results show a novel mechanism for the sensitization to TRAIL-induced apoptosis linking hispidulin treatment to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of death receptor (DR) ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.