The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was highly active in EAC, whereas showing no significant activity in Cox-2-negative cell lines and primary cells isolated from normal mouse esophagus and stomach. Evaluation of infectivity-enhanced vectors revealed that the transduction and virus-cell binding ability of Ad5/Ad3-chimera were significantly more efficient than that of unmodified and Arg-Gly-Asp (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replication and subsequent oncolysis in EAC cells but not in Cox-2-negative cells in vitro, thus confirming the dependence of their replication on the Cox-2 promoter activity. Ad5/Ad3 CRAds exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity. Whereas unmodified and RGD-modified CRAds showed insignificant therapeutic effect in vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of established xenografts in mice. Thus, our studies have demonstrated that Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and potent agents for the treatment of EAC.