AbstractMCR-1 is a lipid A modifying enzyme that confers resistance to the antibiotic colistin. Here, we analyse the impact of MCR-1 expression on E. coli morphology, fitness, competitiveness, immune stimulation and virulence. Increased expression of mcr-1 results in decreased growth rate, cell viability, competitive ability and significant degradation in cell membrane and cytoplasmic structures, compared to expression of catalytically inactive MCR-1 (E246A) or MCR-1 soluble component. Lipopolysaccharide (LPS) extracted from mcr-1 strains induces lower production of IL-6 and TNF, when compared to control LPS. Compared to their parent strains, high-level colistin resistance mutants (HLCRMs) show reduced fitness (relative fitness is 0.41–0.78) and highly attenuated virulence in a Galleria mellonella infection model. Furthermore, HLCRMs are more susceptible to most antibiotics than their respective parent strains. Our results show that the bacterium is challenged to find a delicate equilibrium between expression of MCR-1-mediated colistin resistance and minimalizing toxicity and thus ensuring cell survival.