The prognosis of non-small cell lung cancer (NSCLC) is disappointing because disease recurrence and distant metastasis inevitably occurred. The aim of the present study is to identify novel biomarkers predicting tumor recurrence and metastasis. The 14-3-3ζ protein has been extensively described as a tumor promoter in a panel of solid tumors, including NSCLC. However, there is a big gap regarding the knowledge between 14-3-3ζ and NSCLC recurrence. In this study, we found that overexpression of 14-3-3ζ was more frequent in NSCLC tumor tissues with tumor recurrence. By using scratch healing assay and transwell assay, we demonstrated that NSCLC cells with high expression of 14-3-3ζ gained increased motile and invasive capacity, whereas siRNA-mediated knockdown of endogenous 14-3-3ζ abrogated cancer cell dissemination. Intriguingly, we found that NSCLC cells underwent epithelial-mesenchymal transition (EMT) after the induction of 14-3-3ζ in vitro and in vivo. These findings could be readily recaptured in clinical setting since we showed that NSCLC tumor specimen with high expression of 14-3-3ζ revealed biological features of EMT. Overexpression of 14-3-3ζ also enhanced the phosphorylation of Akt and promoted the proliferation of NSCLC cell lines. In agreement with this notion, we reported that NSCLC cells with high expression of 14-3-3ζ became resistant to chemotherapy-induced apoptosis. These findings strongly suggested that 14-3-3ζ as a novel biomarker predicting risks of disease recurrence and screening 14-3-3ζ status may be a promising approach to select patients who experienced high risks of cancer recurrence and resistance to chemotherapy.