Reactive oxygen species (ROS) have been widely accepted as critical molecules playing regulatory roles in various biological processes, including proliferation, differentiation and apoptotic/ferroptotic/necrotic cell death. Emerging evidence suggests that ROS may be involved in the induction of epithelial-to-mesenchymal transition (EMT), which has been reported to promote cancer stem-like cell (CSC) generation. Recent data indicate that altered accumulation of ROS is associated with CSC generation, EMT and hypoxia exposure, but the underlying mechanisms are poorly understood. In the present study, we derived CSCs from Panc-1 human pancreatic cancer cells and characterized them using serial replating assays and western blot analysis. Functional identification of viable cells was performed using the CCK-8 assay and colony formation assays. The expression of various antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX), was measured by western blot analysis in Panc-1 CSCs. The role of GPX4 in regulating biological processes of Panc-1 CSCs was assessed by proliferation, sphere formation and invasion assays with or without oxidative stress. Manipulation of GPX4 expression by siRNA knockdown or an overexpression vector was performed to assess functions including proliferation, colony formation and invasion. EMT hallmark genes were detected after GPX4 alteration by RT-qPCR and western blot analysis. Panc-1 CSCs displayed more resistance to hypoxia exposure. Compared with the parental Panc-1 cells, Panc-1 CSCs expressed an obviously higher endogenous GPX4 level, indicating their role in maintaining homeostasis. During GPX4 knockdown, ROS accumulation was promoted following oxidative stress exposure to either H 2 O 2 or erastin. Additionally, overexpression of GPX4 eliminated ROS induction by oxidative stress exposure and thus, exerted protective effects on physiological processes in the Panc-1 CSCs. Knockdown of GPX4 arrested cell cycle progression at the G 1 /G 0 phase; inhibited cell proliferation, colony formation, invasion and the stemness phenotype in the Panc-1 CSCs; and decreased the EMT phenotype. Collectively, GPX4 plays a critical role in maintaining oxidative homeostasis and regulates several biological processes, including stemness and EMT, in Panc-1 CSCs.