Background: Activation of glycogen synthase kinase-3 (GSK-3) is involved in the regulation of cell growth, differentiation, mobility, proliferation and survival. However, its clinicopathologic significance remains unclear in prostate cancer (PCa). Materials and Methods: A tissue microarray was produced from 640 samples. Sections were immunostained with an antibody against the non-phosphorylated form of GSK-3(GSK-3 beta) and were digitized. Spearman correlation test was processed for correlations between GSK-3 and biological and clinicopathological variables. The prognostic value of GSK-3 beta was analyzed by Cox Regression model. Results: Cytoplasmic GSK-3 beta was higher in PCa than in normal prostate (mean expression index 4.55 vs. 3.50, p<0.0001). Conversely, nuclear expression was higher in normal prostate than that in PCa (3.38 vs. 2.04, p<0.0001). Cytoplasmic levels of GSK-3 beta were correlated with clinical stage (rho=0.095, p=0.0337), lymph node metastasis (rho=0.116, p=0.0096), extracapsular extension (rho=0.092, p=0.0392), and Gleason score (rho=0.167, p=0.0002). Increased cytoplasmic GSK-3 beta expression was correlated with high Ki-67 labeling index (rho=0.319, p<0.0001), low apoptotic index by TUNEL (rho=-0.118, p=0.0134), high levels of androgen receptor (rho=0.292, p<0.0001) and p-Akt (rho=0.396, p<0.0001). Patients with higher cytoplasmic levels of GSK-3 beta had a twofold risk of biochemical recurrence-free survival compared to those with lower levels of GSK-3 beta [HR 1.934 (1.020-3.667), p=0.043]. Conclusion: Cytoplasmic accumulation of GSK-3 beta is potentially associated with a pro-survival mechanism that promotes PCa development and progression.