Esophageal cancer is one of the most common digestive system neoplasms and has a quite poor prognosis. TNF-related apoptosis-inducing ligand (TRAIL) induces the apoptosis in a wide range of cancer cells including esophageal cancers. However, TRAIL also activates apoptotic pathway in normal cells. To improve the specificity of TRAIL action, we employed the microRNA (miRNA) response elements (MREs) of miR-143 and miR-122 to restrict TRAIL expression mediated by an adenoviral vector (Ad-TRAIL-143-122) in esophageal cancer cells. The experiments showed that Ad-TRAIL-143-122 was able to highly express TRAIL in esophageal cancer cells, but not normal cells. The selective TRAIL expression also led to selective apoptosis in esophageal cancer cells. Ad-TRAIL-143-122 greatly reduced the viability of esophageal cancer cells without cytotoxicity to normal cells. In mice, Ad-TRAIL-143-122 suppressed the growth of esophageal cancer xenografts and protected liver from TRAIL-induced toxicity. In this study, we constructed a biologic vector that can express exogenous genes in a tumor-specific manner. This strategy can simultaneously treat cancer and prevent hepatoxicity and thus may be a promising way for esophageal cancer treatment.