Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.