Iron is an essential element for most cellular processes and recent evidence highlighted its role in regulating the function of hematopoietic stem cells (HSCs). Abnormal iron levels impact HSC quiescence and self-renewal, however, the mechanism by which iron overload (IO) influences HSC function is still unknown. Here, we show that intracellular IO impairs mitochondrial fitness and bioenergetics, inducing metabolic rewiring. In thalassemic mice, as a model of chronic IO, HSCs accumulate mitochondria with elevated reactive oxygen species (mtROS), low membrane potential and reduced oxidative phosphorylation (OXPHOS). Mitochondrial defects are confirmed in other two models of IO, sickle cell disease and iron-loaded wild-type mice, and in vivo iron reduction rescues HSC mitochondria. IO HSCs are highly proliferating and in presence of damaged mitochondria rely on glycolysis for energy production. Notably, restoration of mitochondrial function by targeting in vivo mtROS improved the quiescence and self-renewal of IO HSCs. Our results unravel the critical interplay between iron, ROS and mitochondrial activity in HSCs, revealing that IO shapes HSC metabolic programs. Copyright © 2026 American Society of Hematology.