Embryonic stem (ES) cells are pluripotent cells that are capable of giving rise to any type of cells in the body and possess unlimited self-renewal potential. However, the exact regulatory mechanisms that govern the self-renewal ability of ES cells remain elusive. To understand the immediate early events during ES cell differentiation, we performed a proteomics study and analyzed the proteomic difference in murine ES cells before and after a 6-h spontaneous differentiation. We found that the expression level of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme, is dramatically decreased upon the differentiation. Both knockdown of GPx-1 expression with shRNA and inhibiting GPx-1 activity by inhibitor led to the differentiation of ES cells. Furthermore, we showed that during early differentiation, the quick degradation of GPx-1 was mediated by proteasome. Thus, our data indicated that GPx-1 is a key regulator of self-renewal of murine embryonic stem cells. (C) 2014 Elsevier Inc. All rights reserved.