Nowadays cancer is seriously threat to humans life and heath.Lung cancer is one of most malignant tumors worldwide.Genetic disorder is considered the main reason to be responsible for the happiness of cancer, which causes a high light on gene therapy.Oncolytic viruses is known as a ideal vector to carry anti-tumor gene into host cells due to its high expression and strong stability, especially their ability of selectively lysing cancer cells but not in normal cells after modification.In our study, we replace the endogenous promoter of E1A which is key element for adenovirus replication with survivin promoter, a tumor specific promoter.Meanwhile, the 24bp DNA sequence located on E1A CR2 region are deleted to construct a novel oncolytic adenovirus named Ad.sp-E1A(i÷24).TSLC1 (tumor suppressor in lung cancer 1) is a novel tumor suppressor gene, whose expression was loss in many human cancers, while not in normal cells, It plays an important role in tumor formation, cell adhesion, immune surveillance, cell motility and signal transduction, So TSLC1 is expected to become a potential cancer therapeutic genes.In this study TSLC1 was inserted into the dual-regulated oncolytic adenovirus vector Ad.sp-E1 A(△24), whose endogenous E 1 A promoter was replaced by survivin promoter and E1A 24bp gene were deleted.The novel oncolytic adenovirus Ad.sp-E1A(△24)-TSLC1 exhibited an excellent antitumor effect on lung carcinoma cell growth in vitro but does no or little damage to normal lung cell lines.Real-time PCR analysis demonstrates TSLC 1 gene expression in lung cancer cells was significantly lower than in normal lung cells.Western Blot and Real-time PCR assay indicated that oncolytic adenovirus Ad.sp-E1A(△24)-TSLC1 can selectively replicate in lung cancer cells but not in normal cells.MTT assay displayed a strong inhibitory effect on lung cancer cells growth and crystal violet staining assay showed significant cytopathic effect in lung cancer cells infected with Ad.sp-E1A(△24)-TSLC1.Hochest33342 staining assay was performed to detect apoptotic morphological changes and the underlying mechanism was explored by Western Blot, the results indicated that a(c)tivation of caspase pathway and cleavage of caspase-3, caspase-8, and poly (ADP-ribose) polymerase (PARP)were involved in the apoptosis progress.In conclusion, Oncolytic adenovirus mediating Survivin promoter harboring with TSLC1 Ad.sp-E1A(△24)-TSLC1 is a potent antitumoral agent for clinical therapy of lung cancer and provides a more safe and effective reference for CTGVT.