PatAB is an ABC bacterial transporter that facilitates the export of antibiotics and dyes. The overexpression of genes conferring efflux-mediated fluoroquinolone resistance has been observed in several laboratory strains and clinical isolates of . Using transformation and whole-genome sequencing, we characterized the fluoroquinolone-resistance mechanism of one clinical isolate without mutations in the DNA topoisomerase genes. We identified the PatAB fluoroquinolone efflux-pump as the mechanism conferring a low-level resistance to ciprofloxacin (8 µg/mL) and levofloxacin (4 µg/mL). Genetic transformation experiments with different amplimers revealed that the entire plus the 5'-terminus of are required for levofloxacin-efflux. By contrast, only the upstream region of the operon, plus the region coding the N-terminus of PatA containing the G39D, T43A, V48A and D100N amino acid changes, are sufficient to confer a ciprofloxacin-efflux phenotype, thus suggesting differences between fluoroquinolones in their binding and/or translocation pathways. In addition, we identified a novel single mutation responsible for the constitutive and ciprofloxacin-inducible upregulation of . This mutation is predicted to destabilize the putative rho-independent transcriptional terminator located upstream of , increasing transcription of downstream genes. This is the first report demonstrating the role of the PatAB transporter in levofloxacin-efflux in a pneumoccocal clinical isolate.