Abstract: Objective To explore the effects of infection with space mutagenic E.coli on secretion of in flammatory cytokines and NF-κB expression in RAW264.7 macrophages under simulated microgravity. Methods RAW264.7 macrophages were randomly divided into four groups as Control group(Con), Conrol+T1-13 group(Con+T1-13), Simulated microgravity group (SM) and Simulated microgravity + T1-13 group(SM+T1-13), in which T1-13 is space mutagenic E.coli. A clinostat was used to simulate microgravity on cultured macrophages, then RAW264.7 macrophages were co-cultured with T1-13 E.coli in Con+T1-13 and SM+T1-13 groups for 6 hours. The mRNA expression of TNF-α, IL-1β, IL-6 and NF-κB p65 in macrophages were determined by RT-qPCR, the cytokines concentrations of TNF-α, IL-1β and IL-6 in culture medium were determined by multiplex immunoassay of Luminex xMAP platform, and the total Nitric Oxide Assay Kit was used to detect total NO in cell culture medium. Western Blot was used to detect the protein expression of NF-κB pathway related molecules. Results Compared with Con group, TNF-α, IL-1β and IL-6 concentration in SM group increased by 168%, 77.6% and 187%, respectively. While compared with Con+T1-13 group, TNF-α, IL-1β and IL-6 concentration in SM+T1-13 group only increased by 1.06%,6.78% and 27.1%, respectively. After infected by space mutagenic E.coli, the NF-κB p65 mRNA expression in RAW264.7 increased significantly. Interestingly, the protein expression of IKKβ, IKKα and Phospho-NF-κB p65 and NF-κB p65 decreased significantly. Conclusion Simulated microgravity induces the increased cytokine secretion in macrophages, which canbe inhibited by infection of space mutagenic E.coli. Such inhibition effects might be related with decreased protein expression of NF-κB.