Introduction: Runt-domain transcription factor (RUNX3) has an important role in gene regulation in development. In several cancers, such as gastric cancer, RUNX3 showed tumor suppressive function. Frequent inactivation of RUNX3 by methylation also was found in lung cancer, especially adenocarcinoma. Interestingly, lung adenocarcinoma is observed in RUNX3 heterozygous mice. Recently, inactivation of RUNX3 draws an attention as a potential marker of tumor stem cell. Materials and Methods: We constructed a recombinant adenovirus expressing RUNX3 (ad-RUNX3) by homologous recombination and an adenovirus expressing RUNX3 dominant negative form (ad-RUNX3 DNF) as a nonfunctioning control adenovirus. We tested the function of these adenoviruses in several lung cancer cell lines. Results: Transduction of ad-RUNX3 in lung cancer cell lines showed the production of RUNX3 protein and the expression of RUNX3 was enhanced by vorinostat, histone deacetylase inhibitor which could increase the transduction and transcription of adenoviral transgene. Transduction of ad-RUNX3 showed moderate antitumor effect in lung cancer cell lines. However, combined treatment of ad-RUNX3 with vorinostat showed strong synergistic antitumor effect with enhanced induction of apoptosis. Conclusion: Restoration of RUNX3 via adenoviral vector demonstrated antitumor effect. Further study on ad-RUNX3 will elucidate the role of RUNX3 in lung tumorigenesis.