Objective To detect the expression of phosphatase of regenerating liver (PRL)-3 in primary gastric cancer tissues, evaluate its prognostic impact, and investigate the role of silencing PRL-3 expression by miRNA interference in gastric cancer growth. Methods Immunohistochemistry was used to measure the expression of PRL-3 in 137 gastric tumor samples. The overall survival rates of the patients with different PRL-3 expression levels were compared. Recombinant lentivirus expressing artificial PRL-3 miRNA, Lent. rPRL3-miRs, was established. Human gastric cancer ceils of the line SGC7901 were cultured and transfected with Lent. rPRL3-miRs or blank vector, Lenti. rPRL-miR-neg, respectively. Un-transfected progenitor cells were used as controls. MTT assay was used to examine the proliferation of these cells. RTPCR and Western blotting were used to detect the RNA and protein expression of PRL-3 in the SGC7901 cells. Thirty BALB/c mice were divided into 3 equal groups to be inoculated subcutaneously with SGC7901 ceils transfected with Lent. rPRL3-miRs or blank vector, and control SGC7901 ceils respectively. The growth of tumor was observed and the tumor sizes were measured 21 days later. Results 85 of the 137 gastric cancer samples (62%) showed high PRL-3 expression and 26 (19%) showed moderate and low PRL-3 expression. High PRL-3 expression was significantly correlated with tumor size ( P< 0. 01 ), infiltration depth (P< 0. 01 ), lymph node metastasis ( P< 0. 01 ), hepatic metastasis ( P< 0. 01 ), adjacent organ invasion ( P< 0. 01 ), and TNM staging ( P< 0. 01 ). The median survival time of the patients with high PRL-3 expression in the primary tumor was 18.9 months, significantly shorter than those with moderate or low expression (39.1 and 74.3 months respectively, both P< 0. 01 ). The growth rate of the SGC7901 cells transfected with the recombinant lentivirus expressing artificial PRL-3 miRNA was significantly lower than that of the blank vector-transfected group. The implanted tumor size of the Lenti. rPRL-3-miR-B transfection group was ( 1.92 ±0. 18 ) cm3, significantly smaller than those of the control and Lenti. rPRL-miR-neg groups [(4.86±0.38) and(4.74±0.39)cm3 respectively, both P<0.01]. Conclusion High PRL-3 expression is associated with gastric cancer progression. Silencing of PRL-3 significantly suppresses the proliferation of gastric cancer cells and tumor growth. PRL-3 plays a key role in the growth of gastric cancer. PRL-3 should be considered as a potential therapeutic target.