Ionophores are antibacterial compounds that affect bacterial growth bychanging intracellular concentrations of the essential cations, sodium and potassium.They are extensively used in animal husbandry to increase productivity and reduceinfectious diseases, but our understanding of the potential for and effects of resistancedevelopment to ionophores is poorly known. Thus, given their widespread global usage,it is important to determine the potential negative consequences of ionophore use onhuman and animal health. In this study, we demonstrate that exposure to the ionophoremonensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates invitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistantmutants show that the resistance phenotype is associated with de-repression of de novopurine synthesis, which could be achieved through mutations in different transcriptionalregulators including mutations in the gene purR, the repressor of the purine de novosynthesis pathway. This study shows that mutants with reduced susceptibility to theionophore monensin can be readily selected and highlights an unexplored link betweenionophore resistance, purine metabolism, and fitness in pathogenic bacteria.