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High PRL-3 expression had been reported to have close association with lymph node metastasis (LNM) of gastric cancer. However, the prognostic significance of highly expressing PRL-3 in LNM of human gastric cancer and the role in the metastasis remain unclear. Our study examined PRL-3 expression both in the LNM (n = 107) and in the primary lesion (n = 137) of gastric cancer, and compared the overall survival rates. RNA interference, induced by recombinant plasmid pcDNA.rPRL3-miR expressing artificial PRL-3 miRNA, was employed to knockdown PRL-3 expression in human SGC7901 gastric cancer cells. Invasion assay and migration assay in vitro were conducted to determine the role of PRL-3 in the metastasis. The role of PRL-3 in the proliferation of SGC7901 cells and tumor growth were also determined. We observed that high PRL-3 expression was more frequently detected in the LNM than in the matched primary lesion (72.9 vs. 47.7%, p < 0.001). Furthermore, the overall survival rate of the patients with high expression of PRL-3 in the LNM was significantly less than those with moderate/low expression (p = 0.003). Importantly, knockdown of PRL-3 can significantly reduce both invasion and migration potencies of SGC7901 cells (p < 0.001), and significantly suppressed the proliferation of SGC7901 cells and slowed down the tumor growth (p < 0.001). It was concluded that high expression of PRL-3 in the LNM had a negative impact on the prognosis of the patients, and plays important roles in LNM of gastric cancer and the tumor growth, which can be a potential therapeutic target and a prognostic factor. © 2008 Wiley-Liss, Inc.