Aberrant overexpression of the transcription/translation factor Y-box-binding protein-1(YBX1) is associated with non-small cell lung cancer (NSCLC) aggressiveness. Cancer stem cells (CSCs) contribute to the tumorigenesis and metastasis of NSCLC. Hitherto, the mechanism by which YBX1 regulates CSCs and metastasis in NSCLC remains unclear. Here, we demonstrated that YBX1 levels were elevated in NSCLC tissues and cell lines. Enforced expression of YBX1 promoted NSCLC cells invasion, sphere forming ability and ALDH1(+) population. Conversely, reduced YBX1 impaired CSC properties of NSCLC cells in vitro and tumor-initiating frequencies, as well as metastasis in vivo. Importantly, we described a mechanism whereby YBX1 directly promoted NANOG, a transcription factor, transcriptional activation. Depletion of NANOG abolished the enhanced ability of invasion and sphere formation in YBX1 elevated-A549 cells. Collectively, these findings demonstrate a novel role of YBX1 in maintaining the sternness of CSCs and metastasis, unveiling YBX1 as promising therapeutic target for NSCLC treatments. (C) 2017 The Authors. Published by Elsevier Inc.