Gram-negative bacteria such as E. coli use tripartite efflux pumps such as AcrAB-TolC to expel antibiotics and noxious compounds. A key feature of the inner membrane transporter component, AcrB, is a short stretch of residues known as the gate/switch loop that divides the proximal and distal substrate binding pockets. Amino acid substitutions of the gate loop are known to decrease antibiotic resistance conferred by AcrB. Here we present two new AcrB gate loop variants, the first stripped of its bulky side chains, and a second in which the gate loop is removed entirely. By determining the crystal structures of the variant AcrB proteins in the presence and absence of erythromycin and assessing their ability to confer erythromycin tolerance, we demonstrate that the gate loop is important for AcrB export activity but is not required for erythromycin binding.