An adenoviral vector with RGD-modified fibers and expressing the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD) was constructed, and its antitumor activity was evaluated in vitro and in vivo. An in vitro study showed that treatment with Ad/TRAIL-F/RGD elicited a high rate of apoptosis in human pancreatic and colon cancer cell lines that were either susceptible or resistant to conventional adenovectors. In vivo study showed that direct administration of Ad/TRAIL-F/RGD to an orthotopic implantation tumor model established in the pancreatic tails of nu/nu mice significantly suppressed tumor growth: tumors in the animals treated with Ad/TRAIL-F/RGD were approximately eight times smaller than those in animals treated with a control vector. We also evaluated hTERT promoter activity and the effect of Ad/TRAIL-F/RGD on mesenchymal stem cells. Our results showed that transgene expression from the hTERT promoter in human bone marrow mesenchymal stem cells was minimal. No adverse effect was observed in mesenchymal stem cells treated with Ad/TRAIL-F/RGD. Together, our results suggest that Ad/TRAIL-F/RGD could become a potent therapeutic agent for the management of pancreatic cancer.