Intratumoral heterogeneity remains as a major challenge in the treatment resistance of prostate cancer. Understanding the mechanism of prostate cancer heterogeneity is essential for developing effective therapies. In this study, we reported the heterogeneous activation of Wnt/beta-catenin signaling in prostate cancer. We developed a Wnt/beta-catenin signaling reporting system to directly characterize the differences between Wnt/beta-catenin signaling active (GFP(+)) and inactive (GFP(-)) cells. Compared to GFP(-) cells, GFP(+) cells demonstrated cancer stem cell properties with higher colony formation efficiency, slower cell cycle, higher resistance to docetaxel and higher expression of cancer stem cell markers. In addition, we found that Wnt/beta-catenin signaling is negatively correlated with H3K27me3 levels. Further studies demonstrated that Wnt/beta-catenin signaling affected H3K27me3 levels by regulating the expression of KDM6A, one of the H3K27me3 demethylases. H3K27me3 suppressed Wnt/beta-catenin signaling by directly silencing LEF1 promoter. Together, our studies suggest that Wnt/beta-catenin signaling makes a major contribution to prostate cancer heterogeneity and targeting both Wnt/beta-catenin signaling active and inactive populations is essential for developing more effective therapies. (c) 2020 Elsevier Inc. All rights reserved.