Haematopoietic stem cells (HSC) are responsible for the lifelong production of all circulating blood cells. During aging, HSC undergo changes in function and phenotype, including compromised regenerative potential, loss of quiescence and increased metabolic activity, which results in a drastic increase in the HSC pool, skewed myeloid lineage differentiation and ultimately can lead to the development of immunosenescence, anaemia and haematological cancers. Therefore, treatments that rejuvenate aged HSC could have profound societal health benefits. Vitamin B12 (vB12) is an essential cofactor for various cellular processes, but its levels decline with age in many tissues. Despite vB12 deficiency being associated with many age-related diseases, the role of vB12 in regulating HSC during the aging process remains unknown. Herein, we reveal a pronounced cobalt (vB12) deficiency in aged bone marrow cells, together with reduced vB12 receptor expression on aged HSC. Furthermore, in vitro supplementation of vB12 to cultures of HSC demonstrated a direct regulatory role of vB12 in promoting HSC quiescence, a key character of young HSC, whilst suppressing HSC proliferation and differentiation. We therefore administered vB12 to aged mice in vivo. Subsequent phenotypic analysis of stem and progenitors revealed a significant reduction in the HSC and progenitor pool. In addition, vB12 repletion also promoted the restoration of a balanced myeloid-lymphoid progenitor pool in aged mice. Competitive transplantation analysis showed significantly enhanced lymphoid lineage differentiation and reduced myeloid lineage differentiation from transplanted aged vB12 treated HSC compared to aged HSC, and consequently a correcting of the age-associated myeloid-biased lineage distribution. Together, the data demonstrate a critical role for vB12 in HSC rejuvenation, and suggest the potential of vB12 as a promising agent for age-related diseases.