The human telomerase reverse transcriptase (hTERT) promoter can be used for the tumor-specific expression of transgenes in order to induce selective cancer cell death. The hTERT core promoter is active in cancer cells but not in normal cells. To examine whether the combination of TNF-related apoptosis inducing ligand (TRAIL) treatment and cancer cell-selective expression of the TRAIL-death receptor could induce cell death in TRAIL-resistant cancer cells, we generated a death receptor-4 (DR4)-expressing adenovirus (Ad-hTERT-DR4), in which the expression of DR4 is driven by the hTERT promoter. Upon infection, DR4 expression was slightly increased in cancer cell lines, and cell death was observed in TRAIL-resistant cancer cell lines but not in normal human cells when DR4 infection was combined with TRAIL treatment. We also generated an adenovirus that expresses a secretable isoleucine zipper (ILZ)-fused, extracellular portion of TRAIL (Ad-ILZ- TRAIL). In cells infected with Ad-ILZ-TRAIL, TRAIL was expressed, secreted, oligomerized and biologically active in the induction of apoptosis in TRAIL-sensitive cancer cells. When Ad-hTERT-DR4 infected TRAIL-resistant HCE4 cells and Ad-ILZ-TRAIL infected TRAIL-resistant HCE7 cells were co-cultured, cell deaths were evident 24 h after co-culture. Taken together, our results reveal that the combination of TRAIL and cancer cell-specific expression of DR4 has the potential to overcome the resistance of cancer cells to TRAIL without inducing significant cell death in normal cells.