Frequent SPOP mutation defines the molecular feature underlying one of seven sub-types of human prostate cancer (PrCa). However, it remains largely elusive how SPOP functions as a tumor suppressor in PrCa. Here, we report that SPOP suppresses stem cell traits of both embryonic stem cells and PrCa cells through promoting Nanog poly-ubiquitination and subsequent degradation. Mechanistically, Nanog, but not other pluripotency-determining factors including Oct4, Sox2, and Klf4, specifically interacts with SPOP via a conservative degron motif. Importantly, cancer-derived mutations in SPOP or at the Nanog-degron (S68Y) disrupt SPOP-mediated destruction of Nanog, leading to elevated cancer stem cell traits and PrCa progression. Notably, we identify the Pin1 oncoprotein as an upstream Nanog regulator that impairs its recognition by SPOP and thereby stabilizes Nanog. Thus, Pin1 inhibitors promote SPOP-mediated destruction of Nanog, which provides the molecular insight and rationale to use Pin1 inhibitor(s) for targeted therapies of PrCa patients with wild-type SPOP.