Lung cancer is the most common cancer and the most common cause of cancer-related death in the world. Nestin, a class VI intermediate filament, is known to be a cancer stem cell (CSC) marker as well as a neuroepithelial stem cell marker. High expression levels of nestin are reported in several types of cancers including lung, pancreatic and prostate cancers. Nestin is thought to regulate tumor cell proliferation, migration, invasion and CSC properties. Here, we confirmed nestin expression in non-small cell lung cancer (NSCLC): Immunohistochemical analysis in surgical specimens detected nestin protein expression in the cytoplasm of 20 of 48 adenocarcinoma (AD) cases (41.7%) and 25 of 47 squamous cell carcinoma cases (53.2%). Nestin immunoreactivity significantly correlated with not only tumor size and lymph node metastasis in NSCLC, but also poor survival in surgical patients with AD. High and moderate expression levels of nestin were confirmed in several lung AD cell lines including H1975 and PC-3. Nestin inhibition by shRNA decreased proliferation, migration, invasion and sphere formation in AD cells. Correspondingly, nestin upregulation by nestin gene transfection resulted in the opposite changes. Moreover, Akt inhibitor IV effectively decreased nestin expression via SRY-box containing protein 2 (Sox2) downregulation and overcame the enhanced sphere formation induced by nestin upregulation. Overall, our results show that nestin correlates with the aggressiveness and stemness of AD. Regulation of nestin via Akt/Sox2 is, thus, a promising candidate for novel therapeutic approaches to eradicate CSCs in lung AD.