Objective To observe the changes of inflammatory reaction in tail-suspension mice after infected by E.coli in spaceflight. Methods 40 C57BL/6 mice were randomly divided into four groups: control group(Con), control+E.coli T1-13 group(Con+T1-13), tail suspension group(TS), tail suspension+E.coli T1-13 group(TS+T1-13). The inflammatory cytokines TNF-α, IL-1β and IL-6 production in plasma and mRNA level in intestinal tissue were detected by ELISA and RT-qPCR, and HE staining was used to represent the morphology changes in small intestine tissue. Results Compared with the control group, the expression of inflammatory cytokines in plasma and intestinal tissue of all experimental groups were increased, and the TS+T1-13 group was most significant(P < 0.01 or P < 0.001). HE staining showed that the small intestine mucosa in the experimental groups were damaged in different degrees, and the damage of TS+T1-13 group was most serious. Conclusions The E.coli from spaceflight increased significantly the expression of inflammatory cytokines in plasma and intestinal tissue from infected tail-suspension mice, and brought more serious damages to the small intestinal mucosal barrier, which suggested that the inflammatory reaction would be increased in tail-suspension mice infected by E.coli from spaceflight.