We report the results of the EcAMSat(Escherichia coliAntimicrobial Satellite) autonomous space flight experiment, investigating the role of σsin the development of antibiotic resistance in uropathogenic E. coli(UPEC) in microgravity (µ-g). The presence of σs, encoded by the rpoSgene, has been shown to increase antibiotic resistance in Earth gravity, but it was unknown if this effect occurs in µ-g. Two strains, wildtype (WT) UPEC and its isogenic ΔrpoSmutant, were grown to stationary phase aboard EcAMSat, an 11-kg small satellite, and in a parallel ground-based control experiment; cell growth rates for the two strains were found to be unaltered by µ-g. After starvation for over 24 h, stationary-phase cells were incubated with three doses of gentamicin (Gm), a common treatment for urinary tract infections (which have been reported in astronauts). Cellular metabolic activity was measured optically using the redox-based indicator alamarBlue (aB): both strains exhibited slower metabolism in µ-g, consistent with results from previous smallsat missions. The results also showed that µ-gdid notenhance UPEC resistance to Gm; in fact, both strains were more susceptible to Gm in µ-g. It was also found, via a second ground-control experiment, that multi-week storage in the payload hardware stressed the cells, potentially obscuring small differential effects of the antibiotic between WT and mutant and/or between µ-gand ground. Overall, results showed that the ∆rpoSmutant was 34–37% less metabolically active than the WT for four different sets of conditions: ground without Gm, ground with Gm; µ-gwithout Gm, µ-gwith Gm. We conclude therefore that the rpoSgene and its downstream products are important therapeutic targets for treating bacterial infections in space, much as they are on the ground.