Lung cancer is one of the most common and serious types of cancer. Till now, the treatment of lung cancer has been unsatisfactory, which is associated with poor prognosis and high mortality. Therefore, there is an urgent requirement to investigate the molecular mechanisms underlying lung tumorigenesis. To study the potential function of miR-10b involved in the regulation of lung tumors, we monitored NSCLC cell behaviour including proliferation, apoptosis and cell cycle using CCK-8 and flow cytometry analysis. Real-time PCR was used to detect the expression levels of miR-10b in 75 NSCLC patients' tissues and Western blot was also used to analyze the expression level of genes correlated with apoptosis in NSCLC cells. miR-10b expression levels were higher in NSCLC tissues compared with an adjacent normal tissue control. Silencing of miR-10b inhibited cancer cell progress by arresting cell cycle progression in the G0/G1 phase and promoted apoptosis in NSCLC cells. Western blot analysis of miR-10b-silenced cells revealed up-regulation of apoptosis-inducing members Fas, FasL, Bax and caspase 3, and down-regulation of apoptosis-inhibiting factors Bcl-2 and PCNA. And, a significant inverse correlation between the level of miR-10b and klotho was observed, which has been demonstrated to be a novel tumor suppressor gene. A further in vivo tumor formation study in nude mice indicated that inhibition of miR-10b in lung cancer cells delayed the progress of tumor formation. These findings indicated that miR-10b might serve as a useful potential target for treatment of NSCLC.