[Objective] To reveal the phenotypic and gene expression change of Escherichia coli after exposure to microgravity. [Methods] Rotary cell culture system (RCCS) was used to simulate microgravity environment. The effect of microgravity on E. coli K12 was estimated by measuring the growth kinetics, acid resistance and biofilm formation. RNA-Seq was applied to detect the change of gene expression under low shear modeled microgravity (LSMMG). [Results] LSMMG weakened the growth and acid resistance of E. coli K12 and strengthened the biofilm formation ability. Twenty of 25 genes related to nutrition metabolism and the only two acid resistance related genes were down regulated under LSMMG condition. [Conclusion] Simulated microgravity would lead to a certain change of phenotype and corresponding genes. The enhanced biofilm formation and cytotoxicity would be the potential threats of space flight.