An elevated level of hypoxia-inducible factor 1 (HIF-1) is common in solid tumors and correlates with poor prognosis. Therefore, targeting of HIF-1 presents an appealing approach for cancer therapy. In this study, we developed an adenoviral vector carrying a fusion of human WW domain-containing oxidoreductase (hWWOX) and the HIF-1alpha oxygen-dependent degradation domain (ODD) under the control of a synthetic human recombinant telomerase reverse transcriptase promoter (hrTRTP). Luciferase reporter assay showed elevated promoter activity of the synthetic hrTRTP in tested tumor cell lines, but not in WI-38, a nontransformed cell line. Furthermore, adenoviral hrTRTP-hWWOX-Linker-ODD (Ad-TWLH) expression induced apoptosis in a variety of human cancer cell lines under hypoxic conditions dose dependently. Importantly, Ad-TWLH injection into xenografts of A549 tumor cells dramatically reduced tumor size in vivo. Western blot and immunohistochemistry assays also confirmed that hWWOX-Linker-ODD fusion protein was expressed in the A549 xenografts. And the protein level in center part was much higher than that of the circumjacent area. In conclusion, our dual-regulated adenovirus specifically induced apoptosis in human cancer cell lines under hypoxic conditions in vitro and repressed ectopic xenograft tumor growth in vivo, thus providing a novel strategy for hypoxia-targeted cancer gene therapy.