Background: Protection of hematopoietic system has become a primary goal in the development of novel medical countermeasures against ionization radiation and radiotherapy. This study was to explore the role of rapamycin in normal tissues against radiation. Materials and Methods: Mice were pretreated with rapamycin by i.p. every other day for five times before 5 Gy or 8.5 Gy γ-ray whole body irradiation. Blood cell counts, HE staining of bone marrow and liver, bone marrow transplantation, CFU of spleen were used to measure the damage of hematopoiesis and extramedullary hemopoietic organs. Regular karyotype analysis and expression of γ-H2AX (by flow cytometry and western blot) were used to measure DNA damage. Rad 50 and DNA Lig 4 expression by western blot were to see the DNA repair ability.  Results: The decrease of red blood cells and platelet induced by radiation were alleviated by pretreatment with rapamycin (d 7,15, p<0.01), and the long-term restoration of white blood cells, lymphocytes and bone marrow were enhanced in rapamycin pretreatment group (d 30,40,70, p<0.05). The transplantation experiment also indicates that the long-term reconstitution in lethally irradiated recipient mice was improved in rapamycin group (p<0.05). The hepatocellular injury by radiation was also reduced and the colony formation numbers of spleen after irradiation was improved in rapamycin group (p<0.05). Karyotype analysis indicates that rapamycin protected bone marrow cells from chromosome mutation. Furthermore, expression of DNA repair proteins Rad 50 and DNA Lig 4 was enhanced and DNA damage marker γ-H2AX was reduced in mice exposed to radiation by rapamycin pretreatment. Conclusion: Rapamycin pretreatment mitigates hematopoietic system from radiation injury in both bone marrow and extramedullary hematopoietic organs by improving genomic stability and increasing survival of hematopoietic stem and progenitor cells (HSPCs).