The Escherichia coli AcrB efflux pump is a key RND-type multidrug resistance pump. This study aimed to identify nonaromatic residues in AcrB that determine macrolide resistance (MR). A hybrid AcrB-615-628MexB (replacing AcrB residues 615-628 with homologous MexB sequence) was constructed in an acrB-overexpressing E. coli strain. MIC assays showed the hybrid specifically reduced MR. Single mutations reintroduced into the hybrid revealed that AcrB residue 616 is critical: glycine (G616) confers MR, while asparagine (N616) (from MexB) reduces MR. Introducing G616N into wild-type AcrB reproduced the MR-sensitive phenotype, whereas I626M had no effect. Conclusion: AcrB position 616 determines MR level—G616 leads to macrolide resistance, N616 to sensitivity, with no impact on nonmacrolide antibiotics.