Abstract Background Mutations that inactivate MarR reduce susceptibility to ciprofloxacin and competitive growth fitness in Escherichia coli. Both phenotypes are caused by overexpression of the MarA regulon, which includes the AcrAB-TolC drug efflux pump. Objectives We asked whether compensatory evolution could reduce the fitness cost of MarR-inactivating mutations without affecting resistance to ciprofloxacin. Methods The cost of overexpressing the AcrAB-TolC efflux pump was measured independently of MarA overexpression. Experimental evolution of MarR-inactive strains was used to select mutants with increased fitness. The acquired mutations were identified and their effects on drug susceptibility were measured. Results Overexpression of the AcrAB-TolC efflux pump was found not to contribute to the fitness cost of MarA regulon overexpression. Fitness-compensatory mutations were selected in marA and lon. The mutations reduced the level of MarA protein thus reducing expression of the MarA regulon. They restored growth fitness but also reduced resistance to ciprofloxacin. Conclusions The fitness cost caused by overexpression of the MarA regulon has multiple contributing factors. Experimental evolution did not identify any single pump-independent cost factor. Instead, efficient fitness compensation occurred only by mechanisms that reduce MarA concentration, which simultaneously reduce the drug resistance phenotype. This resistance/fitness trade-off is a barrier to the successful spread of MarR inactivation mutations in clinical isolates where growth fitness is essential.