[Background] The exploration mission of deep space exploration in China in the next few years will be a “well blowout” development. The impact of microorganisms on space activities will attract more and more attention. However, there are few studies on phenotypic heterogeneity at home and abroad. [Objective] To investigate the effect of low-shear modeled microgravity (LSMMG) and low-shear normal gravity (LSNG) on the large intestine from the perspective of phenotypic heterogeneity Bacillus K12 caused by the impact. [Methods] The continuous culture of Escherichia coli K12 was simulated by a rotating cell culture system to simulate the environment of weightlessness. The phenotypic heterogeneity subtypes were selected from the morphology, color and morphology of the cells. The proliferation rate of different strains, antibiotic resistance, biofilm formation, environmental pressure resistance, and cytotoxicity were measured to assess the effects of low shear stress and simulated weightlessness on E. coli K12. [Results] Four strains with different phenotypic heterogeneity were isolated by continuous culture in rotating cell culture system. Two of them were from simulated weightlessness group (M1, Ma) and the other two were from normal gravity control group (N1, Na). Compared with the original strain (P), the four sub-populations showed significant changes in proliferation rate, biofilm formation, environmental pressure resistance and cytotoxicity, and their antibiotic resistance has no significant changes. [Conclusion] Low shear stress can induce the phenotypic heterogeneity of Escherichia coli in both simulated weightlessness environment and normal gravity environment with low shear stress. Compared with the original strain, the phenotypic heterogeneity strains are differentiated. There is no unifying direction, but we still need to be alert to the changing phenotypes that could be harmful to humans.