Although the roles of soluble CD40 ligand (sCD40L) or the klotho gene in lung cancer cells have been studied, little is known about the functions of klotho combined with sCD40L in lung cancer. The present study was designed to investigate the biological effects of klotho combined with sCD40L on the A549 lung cancer cell line (CD40 positive) and their possible mechanisms. Lung cancer A549 cells were chosen as target cells and CD40 signals were stimulated by soluble CD40 ligand (sCD40L). In this study, we found that klotho, soluble CD40 ligand and their combination can increase cell proliferation inhibition and the apoptosis rate in A549 cells by inhibiting the cell cycle, up-regulating Bax gene expression and (or) down-regulating Bcl-2 gene expression; and their combination has a stronger effect on A549 cell apoptosis and proliferation inhibition compared to klotho or sCD40L alone. These data suggest that the combination of klotho and sCD40L may provide an efficient method to treat non-small cell lung cancer.