This study aimed at clarifying the expression of phosphatase of regenerating liver-3 (PRL-3), one member of protein tyrosine phosphatase (PTP) superfamily, in nasopharyngeal carcinoma (NPC) and its correlation with clinicopathologic features, including the survival of patients with NPC. Real-time PCR and Western blot showed that the expression level of PRL-3 was markedly higher in NPC cell lines than that in the normal nasopharyngeal epithelial cell at both mRNA and protein levels. Immunohistochemical analysis revealed overexpression of PRL-3 in 97 of 174 (55.7%) paraffin-embedded archival NPC biopsies. Statistical analysis showed that PRL-3 expression was positively correlated with N classification (p = 0.033), distant metastasis (M classification, p = 0.048) and clinical stage (p = 0.005) of patients. Patients with higher PRL-3 expression had shorter overall survival time, whereas patients with lower level of PRL-3 had better survival. Multivariate analysis suggested that PRL-3 expression might be an independent prognostic indicator for the survival of patients with NPC. Disruption of endogenous PRL-3 protein through a siRNA knockdown technique was shown to suppress the invasion ability and migration potency of 5-8F and HONE1 cells, substantially. Interestingly, we also found that no significant effect on the proliferation of 5-8F and HONE1 cells was observed after PRL-3 was down-regulated. Our results suggest that PRL-3 protein is a valuable marker for progression of NPC patients. High PRL-3 expression is associated with poor overall survival in patients with NPC.