Tetracycline-controllable expression vectors are widely used for inducible expression in mammalian cells. The limitation of this system is the difficulty in expressing high levels of the chimeric transactivator tTA. In this study, we demonstrate a utility of recombinant adenoviruses for the tetracycline-controllable expression system. Unexpectedly, the original tTA transactivator did not show sufficient regulation of the reporter gene expression driven by the tetracycline-responsive promoter (Tet). By adding the nuclear localization signal on the tTA transactivator (NtTA), we achieved tight regulation and high-level induction of the reporter gene expression. The NtTA driven by various promoters demonstrated strict tetracycline controllability at 1 μg/ml of tetracycline and above. The methodology for adenovirus-mediated inducible gene expression has wide applicability. Controllable expression of cytotoxic viral proteins will be applicable for antiviral vaccine productions and pseudotype viral vector generations.