The growth of malignant tumors is associated with mechanisms of immune escape and inhibition of apoptosis. Livin is a novel member of the inhibitors of apoptosis (IAP) protein family that inhibits cell apoptosis. Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. In this study, we used umbilical cord blood (UCB)-derived dendritic cells (DCs) infected with a recombinant adenovirus encoding the livin gene as a vaccine to activate effector cells such as cytotoxic T lymphocytes (CTLs) to recognize and kill livin-expressing cancer cells in vitro as an improved strategy for overcoming the ability of these cancer cells to escape apoptosis and antitumor immune responses. We employed interferon-γ (IFN-γ) enzyme-linked immunospot assays to confirm that our immunization strategy induced an antigen-specific reaction to livin and flow cytometric analysis of staining with Annexin V and PI to measure the cytotoxic activity of the effector cells against the livin-expressing lung cancer cell lines A549 and H460. Our results show that the recombinant adenovirus was able to promote the maturation of the UCB-derived DCs. This DC vaccine could activate antigen-specific T cells to produce IFN-γ upon recognition of livin peptide in the context of the appropriate HLA molecule. The antigen-specific T cells mediate significant cytotoxicity against the cancer cells, but are unlikely to cause an autoimmune reaction against the human bronchial epithelia cells (HBE), which do not express livin.