Glutathione (GSH), the major non-enzymatic antioxidant, plays a critical role in cellular reactive oxygen species (ROS) neutralization. Moreover, GSH is required for the self-renewal maintenance of human embryonic stem cells (hESCs), and is highly accumulated in undifferentiated cells. Among 8 GSH biosynthesis-related enzymes, we found CHAC2 is highly enriched in undifferentiated hESCs. CHAC2 downregulation in hESCs efficiently decreased the levels of GSH and blocked self-renewal. The self-renewal of sh-CHAC2 cells can be rescued by GSH supplement. CHAC2 downregulation promoted mesoderm differentiation and hampered both teratoma formation and the expression of Nrf2 and glutamate-cysteine ligase (GCL). Notably, CHAC1 knockdown restored the self-renewability of CHAC2-downregulated cells. Although both CHAC1 and CHAC2 purified protein alone showed the catalytic activities to GSH, our data extraordinarily revealed that CHAC2 prevented CHAC1-mediated GSH degradation, which suggests that CHAC2 competes with CHAC1 to maintain GSH homeostasis. This is the first report to demonstrate that CHAC2 is critical for GSH maintenance and the novel roles of the CHAC family in hESC renewal.