Background: PHF21B is newly identified to be involved in the tumor progression; however, its biological role and molecular mechanism in prostate cancer have not been defined. This study is aimed to study the role of PHF21B in the progression of prostate cancer. Methods: Real-time PCR, immunohistochemistry and western blotting analysis were used to determine PHF21B expression in prostate cancer cell lines and clinical specimens. The role of PHF21B in maintaining prostate cancer stem cell-like phenotype was examined by tumor-sphere formation assay and expression levels of stem cell markers. Luciferase reporter assay, western blot analysis, enzyme-linked immunosorbent assay and ChIP assay were used to determine whether PHF21B activates the Wnt/beta-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2. Results: Our results revealed that PHF21B was markedly upregulated in prostate cancer cell lines and tissues. High PHF21B levels predicted poorer recurrence-free survival in prostate cancer patients. Gain-of-function and loss-of-function studies showed that overexpression of PHF21B enhanced, while downregulation suppressed, the cancer stem cell-like phenotype in prostate cancer cells. Xenograft tumor model showed that silencing PHF21B decreased the ability of tumorigenicity in vivo. Notably, Wnt/beta-catenin signaling was hyperactivated in prostate cancer cells overexpressing PHF21B, and mediated PHF21B-induced cancer stem cell-like phenotype. Furthermore, PHF21B suppressed repressors of the Wnt/beta-catenin signaling cascade, including SFRP1 and SFRP2. These results demonstrated that PHF21B constitutively activated wnt/beta-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2, which promotes prostate cancer stem cell-like phenotype. Conclusions: Our results revealed that PHF21B functions as an oncogene in prostate cancer, and may represent a promising prognostic biomarker and an attractive candidate for target therapy of prostate cancer.