In this study, we investigated the resistance mechanisms to fluoroquinolones of 85 non-cystic fibrosis strains of Pseudomonas aeruginosa exhibiting a reduced susceptibility to ciprofloxacin (MICs from 0.25 to 2 μg/ml). In addition to MexAB-OprM (31 of 85 isolates) and MexXY/OprM (39 of 85), the MexEF-OprN efflux pump (10 of 85) was found to be commonly upregulated in this population that is considered susceptible or of intermediate susceptibility to ciprofloxacin, according to current breakpoints. Analysis of the 10 MexEF-OprN overproducers (nfxC mutants) revealed the presence of various mutations in the mexT (2 isolates), mexS (5 isolates), and/or mvaT (2 isolates) genes, the inactivation of which is known to increase the expression of the mexEF-oprN operon in reference strain PAO1-UW. However, these genes were intact in 3 of 10 of the clinical strains. Interestingly, ciprofloxacin at 2 μg/ml or 4 μg/ml preferentially selected nfxC mutants from wild-type clinical strains (n = 10 isolates) and from first-step mutants (n = 10) overexpressing Mex pumps, thus indicating that MexEF-OprN represents a major mechanism by which P. aeruginosa may acquire higher resistance levels to fluoroquinolones. These data support the notion that the nfxC mutants may be more prevalent in the clinical setting than anticipated and strongly suggest the involvement of still unknown genes in the regulation of this efflux system.