AcrAB-TolC is a major tripartite multidrug efflux pump conferring resistance to a wide variety of compounds in Gram-negative pathogens. Many AcrB mutants have been constructed through site-directed mutagenesis to probe the mechanism of AcrB function in antibiotic resistance. However, much less is known about the actual drug resistance related mutants that naturally occur in clinically isolated pathogens. Here, we report two novel AcrB substitutions, M78I and P319L, in clinically isolated strains with high-level ciprofloxacin resistance. Plasmids expressing the detected mutations were constructed and introduced into SL1344△ Antimicrobial susceptibility assay showed that all AcrB M78I, AcrB P319L and AcrB M78I/319L conferred reduced susceptibilities to multiple substrates, including fluoroquinolones, erythromycin, tetracyclines, bile salts and dyes. Site-directed mutagenesis and MIC results revealed that increased hydrophobicity of M78I was one of the reasons why AcrB M78I had lower susceptibility to fluoroquinolones. Fluorescence labeling experiments suggested that the AcrB M78I substitution enhanced the binding of substrates to certain amino acid sites in the efflux pathway (e.g., site Q89, E673 and F617) and weakened the binding to other amino acids (e.g., S134 and N274). Structural modeling disclosed the increased flexibility of Leu was favorable for the functional rotation of AcrB compared to the original Pro. AcrA 319L makes the functional rotation of AcrB more flexible, this enables substrate efflux more efficiently. In order to understand the mechanism of AcrAB-TolC drug efflux well, interaction between AcrA and AcrB in the role of substrate efflux of AcrAB-TolC should be further investigated.