Hematopoietic stem cells (HSC) are maintained and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and non-diabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin and N-cadherin on LT-HSCs, and β1-integrin, β-catenin, angiopoietin-1 and CXCL12 on OPN cells. In an in vitro co-culture system, LT-HSCs isolated from non-diabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to non-diabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin and N-cadherin were restored to normal levels. The effects of diabetic or non-diabetic OPN cells on LT-HSCs shown in this co-culture system were confirmed by the co-injection of LT-HSCs and OPN cells into bone marrow of irradiated non-diabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities, and suggest that the replacement of OPN cells may represent a novel treatment strategy.