Klotho (KL) is a transmembrane protein that can be shed, and act as a circulating hormone and modulate several signaling pathways. There also exists a splice variant of Klotho mRNA, which encodes a putative secreted protein (Klotho-S, KL-S) in both human and mouse. The potential anti-senescence gene Klotho has been recently found to participate in the progression of several different human cancers. In the current study, we undertook to study the expression and activity of Klotho in lung cancer cell line A549. Klotho expression was studied by using RT-PCR and western blotting. Effects of Klotho on cell growth and motility were assessed using MTT and scratch motility assay, and the apoptosis was assessed by TUNEL. Wnt signaling pathway activity was measured by western blotting. We established that the Klotho was endogenous expressed in A549 cells, but the expression level is lower compared with normal lung tissues. The overexpression of KL or KL-S could inhibit the cell proliferation, motility, and induce apoptosis in a dose-dependent manner. Also, we report KL could inhibit activation of Wnt -TCF/β-catenin signaling pathway, and it is involved in KL-induced growth inhibition. These studies indicate Klotho works as a potential tumor suppressor in lung cancer, and suggest that the Klotho tumor suppressive activities could be mediated through its KL-S isoform. These results suggest the use of Klotho or KL-S as potential strategy for the development of novel therapeutic interventions for lung cancers.