Hematopoietic stem cells (HSCs) maintain stemness through various mechanisms that protect against stressful conditions. Heat shock proteins (HSPs) preserve cell homeostasis during stress responses through protein quality control, suggesting that HSPs may safeguard HSCs against numerous traumas. Here, we show that mortalin, a mitochondrial HSP, plays an essential role in maintaining HSC properties by regulating oxidative stress. Mortalin is primarily localized in hematopoietic stem and progenitor cell (HSPC) compartments. In this study, the inhibition of mortalin function caused abnormal reactive oxygen species (ROS) elevation in HSCs and reduced HSC numbers. Knockdown (KD) of mortalin in HSPCs impaired their ability to repopulate and form colonies. Moreover, mortalin-KD HSCs could not maintain quiescence and showed severe downregulation of cyclin-dependent kinase inhibitor- and antioxidant-related genes. Conversely, HSCs that overexpressed mortalin maintained a high reconstitution capacity and low ROS levels. Furthermore, DJ-1, one of the genes responsible for Parkinson's disease, directly bound to mortalin and acted as a negative ROS regulator. Using DJ-1-deficient mice, we demonstrated that mortalin and DJ-1 coordinately maintain normal ROS levels and HSC numbers. Collectively, these results indicate that the mortalin/DJ-1 complex guards against mitochondrial oxidative stress and is indispensable for the maintenance of HSCs.