A major obstacle in the development of effective recombinant adeno-associated virus (rAAV) mediated gene therapy is infection specificity and gene targeting. In the present study, we investigated whether the human telomerase reverse transcriptase (hTERT) promoter could drive tumor-specific expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis apoptosis-inducing protein with potential toxic effects on normal cells. Our data demonstrated that hTERT promoter-driven tumor-specific expression of TRAIL decreased the cellular viability of tumor cells, but not normal cells. TRAIL expression driven by hTERT promoter inhibited tumor growth significantly in vivo and combination of viral infection with 5-fluorouracil (5-Fu) suppressed tumor growth more efficiently. Intra-venous injection of virus showed that the recombinant virus was predominantly distributed in the liver, but not in other major tissues tested, and no transgene expression was detected in the liver. Furthermore, serum enzyme and liver histology analysis confirmed that liver function is unaffected by TRAIL expression, significant as the liver is frequently metastasized and scattered with tumors from other organs, which are unpractical to treat by intra-tumor injection. Together our results demonstrate that rAAV-mediated TRAIL expression is a promising strategy in gene therapy for treatment of cancer.