This study aimed to investigate the properties of mutations of the active efflux pump genes acrA/B and tolC in Escherichia coli CVCC 1547 when induced by different drugs. The mutations were isolated in vitro by exposing E. coli CVCC 1547 to stepwise increases in the concentration of ceftriaxone (CRO), amikacin (AMK), or ciprofloxacin. The results showed that the minimum inhibitory concentrations for the corresponding drugs increased, as did the minimum inhibitory concentrations for other fluoroquinolones and β-lactam drugs that were not inducers. DNA sequence analyses of the acrA/B and tolC genes of the mutants and comparison with the parent strain revealed that genetic variations had occurred. Three point mutations resulted in amino acid changes in the proteins expressed. Specifically, strain CRO10 had a mutation in acrA, A309G, that resulted in a Thr-103 to Ala substitution, and a mutation in tolC, G735A, that changed Ala-245 to Thr; strain AMK20 (and AMK30) had a Val-447 to Ile amino acid change in acrB. In addition to the missense mutations in these strains, we detected 7, 20, and 15 nonsense mutations in acrA, acrB, and tolC, respectively. To sum up, multiple genetic sequence variations and some changes in amino acid sequences were detected when E. coli CVCC 1547 was challenged in vitro with CRO, AMK, or ciprofloxacin. These changes may have given rise to multidrug-resistant strains.