Objective: Previous studies have shown that inhibition of inducible NO synthase (NOS2 or iNOS) with an inhibitor can selectively protect several normal tissues against radiation during radiotherapy. However, the role of NOS2 in ionizing radiation (IR)-induced bone marrow (BM) suppression is unknown and thus was investigated in the present study using NOS2(-)/(-) and wild-type mice 14 days after they were exposed to a sublethal dose of total body irradiation (TBI). Methods: The effects of different doses of IR (1, 2 and 4 Gy) on the apoptosis and colony-forming ability of bone marrow cells from wild-type (WT) and NOS2(-)/(-) mice were investigated in vitro. In addition, we exposed NOS2(-)/(-) mice and WT mice to 6-Gy TBI or sham irradiation. They were euthanized 14 days after TBI for analysis of peripheral blood cell counts and bone marrow cellularity. Colony-forming unit-granulocyte and macrophage, burst-forming unit-erythroid and CFU-granulocyte, erythroid, macrophage in bone marrow cells from the mice were determined to evaluate the function of hematopoietic progenitor cells (HPCs), and the ability of hematopoietic stem cells (HSCs) to self-renew was analysed by the cobblestone area forming cell assay. The cell cycling of HPCs and HSCs were measured by flow cytometry. Results: Exposure to 2 and 4 Gy IR induced bone marrow cell apoptosis and inhibited the proliferation of HPCs in vitro. However, there was no difference between the cells from WT mice and NOS2(-)/(-) mice in response to IR exposure in vitro. Exposure of WT mice and NOS2(-)/(-) mice to 6 Gy TBI decreased the white blood cell, red blood cell, and platelet counts in the peripheral blood and bone marrow mononuclear cells, and reduced the colony-forming ability of HPCs (P < 0.05), damaged the clonogenic function of HSCs. However, these changes were not significantly different in WT and NOS2(-)/(-) mice. Conclusion: These data suggest that IR induces BM suppression in a NOS2-independent manner.