The increased expression of phosphatase of regenerating liver-3 (PRL‑3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL‑3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP‑SF) revealed the constitutive cytoplasmic expression of PRL‑3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL‑3. The AR-negative cell lines exhibited negligible PRL‑3 expression, and the ectopic overexpression of PRL‑3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL‑3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL‑3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL‑3 expression was significantly higher in the malignant cores, as compared to the non‑malignant areas. Increases in both total and nuclear PRL‑3 levels were also associated with a higher GS and GG. Metastatic tumors (T4‑stage) had lower cytoplasmic, but higher nuclear PRL‑3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL‑3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL‑3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL‑3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.