[Background] In recent years, with the continuous development of manned spaceflight, the issue of space microbial safety cannot be ignored. The disease spectrum of manned spaceflight indicates that infectious diseases have great impact on the health of astronauts. Escherichia coli is a normal flora in the human intestinal tract. Meanwhile, it can cause intestinal infection under certain conditions and become a conditional pathogen. It is of great significance to analyze the effect of space environment on the metabolism of E. coli. [Objective] E. coli was carried on the “SJ-10” satellite for 12 days to analyze and identify the changes in the metabolic level of E. coli in the space environment. [Methods] The non-targeted metabolomics technique was used to collect data and perform mass spectrometry analysis from E. coli carried on the satellite. At the same time, a ground control group was set up. Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify potential differential biomarkers between groups. [Results] A total of 12 significant biomarkers were found by analysis and identification. In the spaceflight group, 11 biomarkers involved in energy metabolism and glycerophospholipid metabolism were up-regulated, and one fatty acid content was down-regulated. [Conclusion] Space flight can improve the level of energy metabolism and glycerophospholipid metabolism of E. coli, and the changes of related metabolites suggest that space flight may promote the proliferation of E. coli.