We previously identified JWA as a novel microtubule-associated protein (MAP), which is implicated in carcinogenesis and tumor progression. The aims of the present study were to investigate the biological action and the prognostic significance of JWA in hepatocellular carcinoma (HCC). Quantitative real-time PCR and Western blot were used to detect JWA mRNA and protein expression, respectively, in stepwise metastatic HCC cell lines and HCC tissues. Short hairpin RNA was used to inhibit JWA expression in HCC cells. The effects of JWA depletion on cell migration, invasion, adhesion and in vivo metastasis were investigated. Immunohistochemistry of JWA was conducted in microarrays with tissue from 314 HCC patients who had undergone surgical resection. Prognostic significance was assessed using the Kaplan–Meier method and log-rank tests. The result showed JWA expression was decreased in the highly metastatic HCC cell lines and HCC tissues. Depletion of JWA caused a notable increase in cell migration, invasion and adhesion in vitro and metastasis in vivo. Furthermore, there was an inverse correlation between JWA expression and FAK expression and phosphorylation, RhoA activation and matrix metalloproteinase-2 (MMP-2) activity in HCC cells. More notably, multivariate analysis revealed that a low level of JWA expression was an independent prognosticator for both recurrence-free and overall survival for HCC patients after surgical resection, especially for AFP-normal HCC patients. Taken together, our data demonstrate that JWA plays a crucial role in HCC progression and suggest JWA may be a potential prognostic biomarker and therapeutic target for HCC. © 2012 Wiley Periodicals, Inc.