The homeobox transcription factor Nanog has a vital role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). Stabilization of Nanog proteins is essential for ESCs. The ubiquitin-proteasome pathway mediated by E3 ubiquitin ligases and deubiquitylases is one of the key ways to regulate protein levels and functions. Although ubiquitylation of Nanog catalyzed by the ligase FBXW8 has been demonstrated, the deubiquitylase that maintains the protein levels of Nanog in ESCs yet to be defined. In this study, we identify the ubiquitin-specific peptidase 21 (USP21) as a deubiquitylase for Nanog, but not for Oct4 or Sox2. USP21 interacts with Nanog protein in ESCs in vivo and in vitro. The C-terminal USP domain of USP21 and the C-domain of Nanog are responsible for this interaction. USP21 deubiquitylates the K48-type linkage of the ubiquitin chain of Nanog, stabilizing Nanog. USP21-mediated Nanog stabilization is enhanced in mouse ESCs and this stabilization is required to maintain the pluripotential state of the ESCs. Depletion of USP21 in mouse ESCs leads to Nanog degradation and ESC differentiation. Overall, our results demonstrate that USP21 maintains the stemness of mouse ESCs through deubiquitylating and stabilizing Nanog.