Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), a growth suppressor that negatively regulates CDK2 activity, has been implicated in various types of cancer; yet its role in lung cancer remains unclear. In the present study, a lentivirus-based system was used to specifically downregulate or upregulate CDK2AP1 expression. A549 lung cancer cells were treated with RNAi (RNA interference) or lentiviral vectors for overexpression. Ectopic overexpression of CDK2AP1 in A549 cells in vitro greatly impaired their proliferation and colony-forming ability and enhanced their chemosensitivity to cisplatin and paclitaxel and caused cell cycle arrest at G1/S transition accompanied by the reduction of expression of CDK4 and CDK7. Injection of the ectopically CDK2AP1-overexpressing A549 cells into nude mice resulted in growth arrest of solid lung cancer tumors in vivo. Knockdown of CDK2AP1 in A549 cells, however, gave rise to the opposite effects including promoting cell proliferation/growth, cell cycling in vitro and enhancing tumorigenesis in vivo. These results suggest that CDK2AP1 plays an important role in modulating the growth and tumorigenesis of lung cancer cells and also has significant effects on the chemosensitivity of pulmonary malignancies to chemotherapeutics. Hence, this study extends our knowledge on the relationship between CDK2AP1 and oncogenesis of lung cancer, indicating that CDK2AP1 may serve as a new molecular target for future lung cancer therapy.