The regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in cancer chemotherapy is not fully understood. Here, we show that the histone deacetylase (HDAC) inhibitors induce TRAIL in human breast cancer cells. Induction of TRAIL by the HDAC inhibitor MS275 can be enhanced by Adriamycin. Using different reporter constructs in conjunction with transcription activity assays and chromatin immunoprecipitation assays, we provide evidence that the transcription factor Sp1 is responsible for TRAIL induction by MS275 alone or in combination with Adriamycin. Further, we show that the combined treatment of breast cancer cells with MS275 and Adriamycin significantly increases apoptotic cell death via the activation of both death receptor and mitochondrial apoptotic pathways. Down-regulation of TRAIL by small interfering RNA silencing decreased MS275-mediated Adriamycin-induced caspase activation and apoptosis, thus conferring Adriamycin resistance. More importantly, breast cancer T47D cells in which Sp1 was knocked down or Sp1-knockout mouse embryonic stem cells were resistant to the combined treatments. Taken together, our results indicate that induction of TRAIL by the combined treatments with MS275 and Adriamycin is mediated by Sp1 and suggest that transcription factor Sp1 is an important target for the development of novel anticancer agents.