Hematopoietic stem cells (HSCs) rely on self-renewal to sustain stem cell potential and undergo differentiation to generate mature blood cells. Mitochondrial fatty acid β-oxidation (FAO) is essential for HSC maintenance. However, the role of Carnitine palmitoyl transferase 1a (CPT1A), a key enzyme in FAO, remains unclear in HSCs. Using a Cpt1a hematopoietic specific conditional knock-out (Cpt1aΔ/Δ) mouse model, we found that loss of Cpt1a leads to HSC defects, including loss of HSC quiescence and self-renewal, and increased differentiation. Mechanistically, we find that loss of Cpt1a results in elevated levels of mitochondrial respiratory chain complex components and their activities, as well as increased ATP production, and accumulation of mitochondrial reactive oxygen species (mitoROS) in HSCs. Taken together, this suggests hyperactivation of mitochondria and metabolic rewiring via upregulated glucose-fueled oxidative phosphorylation (OXPHOS). In summary, our findings demonstrate a novel role for Cpt1a in HSC maintenance and provide insight into the regulation of mitochondrial metabolism via control of the balance between FAO and glucose-fueled OXPHOS.