As the duration of spaceflight missions increases, it becomes important to evaluate the long-term effects of microgravity on bacteria. For the first time, we have grown Escherichia coli K-12 (E. coli) strain MG1655 for up to a thousand generations under simulated microgravity conditions (1000G) using an antibiotic based sterilization protocol between cycles. The long-term adapted cells were examined by scanning electron microscopy and subjected to various antibiotic and chemical stresses in order to assess stress response. In addition, molecular studies identified significant changes in gene expression relative to a short-term adapted control and established that multiple mutations had occurred. The results provide significant evidence that the initial environmental response to microgravity seen in short-term studies is in fact replaced by a different long-term adaptation that is based at least in part on genomic changes. The protein Hfq did not appear to be involved in the adaptation. A portion of the long-term adaptation related to antibiotic resistance, may in part be a response to the sterilization protocol used.