Escherichia coliis a facultative anaerobe found in a wide range of environments. Commonly described as the laboratory workhorse,E. coliis one of the best characterized bacterial species to date, however much of our understanding comes from studies involving the laboratory strainE. coliK-12. Resistance-nodulation-division efflux pumps are found in Gram-negative bacteria and can export a diverse range of substrates, including antibiotics.E. coliK-12 has six RND pumps; AcrB, AcrD, AcrF, CusA, MdtBC and MdtF, and it is frequently reported that allE. colistrains possess these six pumps. However, this is not true ofE. coliST11, a lineage ofE. coli, which is primarily composed of the highly virulent important human pathogen,E. coliO157:H7. Here we show thatacrFis absent from the pangenome of ST11 and that this lineage ofE. colihas a highly conserved insertion within theacrFgene, which when translated encodes 13 amino acids and two stop codons. This insertion was found to be present in 97.59 % of 1787 ST11 genome assemblies. Non-function of AcrF in ST11 was confirmed in the laboratory as complementation withacrFfrom ST11 was unable to restore AcrF function inE. coliK-12 substr. MG1655 ΔacrBΔacrF. This shows that the complement of RND efflux pumps present in laboratory bacterial strains may not reflect the situation in virulent strains of bacterial pathogens.