Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body, thus holding great promise as a renewable source of cells for human therapy. The mechanisms that maintain self-renewal of ESCs remain unclear. Here we show that Nanog, a transcription factor crucial for the self-renewal of ESCs, is phosphorylated at multiple Ser/Thr-Pro motifs. This phosphorylation promotes the interaction between Nanog and the prolyl isomerase Pin1, leading to Nanog stabilization by suppressing its ubiquitination. Inhibition of Pin1 activity or disruption of Pin1–Nanog interaction in ESCs suppresses their capability to self-renew and to form teratomas in immunodeficient mice. Therefore, in addition to the stringent transcriptional regulation of Nanog, the expression level of Nanog is also modulated by posttranslational mechanisms.