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Abstract Investigating the evolution ofEscherichia coliin microgravity offers valuable insights into microbial adaptation to extreme environments. Here the effects of simulated microgravity (SµG) on gene expression and genome evolution ofE. coliREL606, a strain evolved terrestrially for 35 years, is explored. The transcriptomic changes for glucose-limited and glucose-replete conditions over 24 h illustrate that SµG increased the expression of genes involved in stress response, biofilm, and metabolism. A greater number of differentially expressed genes related to the general stress response (GSR) and biofilm formation is observed in simulated microgravity cultures under glucose-limited conditions in comparison to glucose-replete conditions. Longer term SµG culture under glucose-limited conditions led to the accumulation of unique mutations when compared to control cultures, particularly in themraZ/fruRintergenic region and theelyC gene, suggesting changes in peptidoglycan and enterobacterial common antigen (ECA) production. These findings highlight the physiological and genomic adaptations ofE. colito microgravity, offering a foundation for future research into the long-term effects of space conditions on bacterial evolution.