Objectives The worldwide rapid increase in antibiotic-resistant bacteria has made efforts to prolong the lifespan of existing antibiotics very important. Antibiotic resistance often confers a fitness cost in the bacterium. Resistance may thus be reversible if antibiotic use is discontinued or reduced. To examine this concept, we performed a 24 month voluntary restriction on the use of trimethoprim-containing drugs in Kronoberg County, Sweden. Methods The intervention was performed on a 14 year baseline of monthly data on trimethoprim resistance and consumption. A three-parameter mathematical model was used to analyse the intervention effect. The prerequisites for reversion of resistance (i.e. fitness cost, associated resistance and clonal composition) were studied on subsets of consecutively collected Escherichia coli from urinary tract infections. Results The use of trimethoprim-containing drugs decreased by 85% during the intervention. A marginal but statistically significant effect on the increase in trimethoprim resistance was registered. There was no change in the clonal composition of E. coli and there was no measurable fitness cost associated with trimethoprim resistance in clinical isolates. The frequency of associated antibiotic resistances in trimethoprim-resistant isolates was high. Conclusions A lack of detectable fitness cost of trimethoprim resistance in vitro together with a strong co-selection of other antibiotics could explain the rather disappointing effect of the intervention. The result emphasizes the low possibility of reverting antibiotic resistance once established and the urgent need for the development of new antibacterial agents.