The apoptotic ligand TNF-related apoptosis-inducing ligand (TRAIL) is believed to be a promising candidate for cancer gene therapy, yet gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumor tissue. Mesenchymal stem cells (MSCs) have been shown to home to tumor sites and could potentially act as a shield and vehicle for an antitumor gene therapy vector. Here, we used an adenoviral vector expressing TRAIL to transduce MSCs and studied the apoptosis-inducing activity of these TRAIL-carrying MSCs on esophageal cancer cell Eca-109. Our results showed that, in vitro, TRAIL-expressing MSCs were able to inhibit proliferation and induce apoptosis in Eca-109 cells by an MTT assay, co-culture experiments and flow cytometry analysis. In vivo, TRAIL-expressing MSCs also displayed an ability to inhibit tumor growth in an Eca-109 xenograft mouse model. Together, our findings indicated that the gene therapy strategy of MSCs-based TRAIL gene delivery has a wide potential value for improving the treatment of esophageal cancer.