At present, there have been no systematic in-depth in vivo studies that reporting the influence of hepatitis B virus (HBV) on hematopoietic systems, especially its influence on hematopoietic stem cells. Here we use an acute HBV-infected mouse model, established by hydrodynamic injection, to show that hematopoietic stem cells (HSCs) isolated from HBV-infected mice were slightly infected with HBV on day 7 of infection, but they were clear on day 20. There was also a transient decrease in the populations of HSCs, multipotent progenitors (Lin-, c-kit+, Sca1+), and oligopotent progenitors (Lin-, c-kit+, Sca1-) in the bone marrow of infected mice. The influence of HBV on proliferation and colony-formation of MNCs also was assessed. The proliferation of MNCs was significant on day 7, when the virus titer was the highest. The number of colony-forming unit-erythroid (CFU-E) cells and colony-forming unit granulocyte-macrophage (CFU-GM) cells also were significantly inhibited in the mononuclear cells (MNCs) in HBV-infected mice. Further, we analyzed the secretion of erythropoietin (EPO) and granulocyte-macrophage colony stimulating factor (GM-CSF) in bone marrow MNCs and peripheral blood serum. The result showed that the expression of EPO in bone marrow MNCs declined but there was a compensatory increase in peripheral blood serum. No expression of GM-CSF could be detected in bone marrow MNCs, and there was no change to GM-CSF in peripheral blood serum. However, despite infection with HBV, the absolute numbers of white blood cells, neutrophils, monocytes, lymphocytes, and red blood cells in peripheral blood remained stable. These results may provide a basis to study the effects of HBV on hematopoiesis in vivo and identify the mechanism whereby HBV infection affects hematopoiesis. (Am J Transl Med 2017. 1:42-56).