The 14-3-3 protein has been shown to be involved in the cancer process. However, there is no understanding of the relationship between 14-3-3γ (14-3-3 gamma) expression and prognosis in advanced non-small cell lung cancer. In this study, we therefore investigated the association between protein levels by immunohistochemistry and clinicopathological features of advanced NSCLC patients. Survival curves were estimated using the Kaplan-Meier method and tested by log-rank. Multivariate analysis was conducted with the Cox's regression model to determine independence of factors. p values less than 0.05 were considered significant. A total 153 patients were studied, with 54.3% being stage III and 45.8% stage IV. Fifty-one cases (33.3%) were squamous cell carcinomas, and 98 cases (64.1%) were adenocarcinomas. High 14-3-3γ expression was seen in 59.5% and significantly correlated with lymph node metastasis (p=0.010) and distant metastasis (p=0.017). On Kaplan-Meier analysis, high 14-3-3γ expression was associated with poorer survival with a marginal trend toward significance (p=0.055). On multivariate analysis, age, treatment, and 14-3-3γ expression proved to be independent prognostic parameters. In vitro experiments indicated that 14-3-3γ overexpression also played a potential role in cancer invasion. In conclusion, our data suggest that 14-3-3γ overexpression is associated with invasion and a poor prognosis. Therefore, 14-3-3γ may be a potential prognostic marker of advanced non-small cell lung cancer.