Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentration-dependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival. Copyright © 2019 Elsevier Inc. All rights reserved.