Tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor superfamily that induces apoptosis in various cancer cell types but not in most normal cells. However, it is clear that not all cancer cells are sensitive to the killing effects of TRAIL, including breast cancer. Previous studies have demonstrated that chemotherapeutic drugs sensitize tumor cells to apoptosis induced by TRAIL in several types of malignancies. In the present study, we studied the effects of TRAIL combined with cisplatin on two breast cancer cell lines MDA-MB-468 and HCC-1937 in vitro. MTT assay, crystal violet staining assay, DAPI staining assay and flow cytometric analysis were undertaken to evaluate the enhancement of breast cancer cell death using Ad-sTRAIL and/or cisplatin. The levels of apoptotic molecules in signal transduction pathways were analyzed by real-time RT-PCR and western blotting. We found that co-treatment with Ad-sTRAIL and cisplatin exhibited stronger cytotoxicity and induced more significant apoptosis in breast cancer cells compared with Ad-sTRAIL or cisplatin alone. Pretreatment with cisplatin significantly enhanced the expression of DR5. Moreover, the induction of apoptosis by TRAIL plus cisplatin was accompanied by the downregulation of cFLIP and BCL2L1, and simultaneously robust enzymatic activation of caspase-8, culminating in decreased cancer cell survival. The present study revealed that TRAIL conjugated with cisplatin exhibited a markedly increased cytotoxic and apoptosis-inducing effect on breast cancer cells.