Resistance of Salmonella spp to antimicrobials of the fluoroquinolone (FQ) resistance is increasing worldwide. Among the mechanisms involved, increased efflux via the tri-partite AcrAB-TolC multidrug efflux system is mainly transcriptionally controlled by the ramRA regulatory locus. The binding of RamA to the operator regions of the acrAB and tolC genes activates their transcription. In susceptible strains, ramA transcription is repressed by RamR, whose gene is located immediately upstream of ramA. RamA overproduction associated to mutations that alter the RamR repressor or its binding to the ramA promoter classically mediate a two- to four-fold increase of resistance levels in clinical and experimentally selected strains of S. Typhimurium and other S. enterica serovars. Furthermore, a recent study showed that ramA overexpression could decrease the expression of cell invasion-related genes of the type III secretion system-1 (T3SS-1). A matter of concern is that ramRA mutations resulting in enhanced efflux-mediated resistance to multiple antibiotics including FQ, may also influence the virulence of clinical strains. In this study, we used qRT-PCR and cell invasion assays to address the effect on the transcription of effluxand invasion-related genes and on the invasiveness, of natural ramRA mutations present in three FQresistant S. Typhimurium clinical isolates of the DT104 and DT204 epidemic clones. As previously shown, the investigated mutations altering the RamR repressor or its DNA binding site increased expression of efflux genes dependently on ramA. However, the decreased expression of T3SS-1 genes previously reported was not always observed and seemed to be dependent on the genetic background of the FQ-resistant isolate. As well a ramA-dependent decreased invasion of intestinal epithelial cells was observed for only one particular clinical ramR mutant. In conclusion, the regulation of S. Typhimurium invasiveness by the ramRA locus appears variable both at the transcriptional and at the phenotypic level, even between strains of the same definitive phage type. Therefore, the acquisition of natural ramRA mutations that confer increased resistance levels to multiple antibiotics and participate in FQ-resistance may also negatively modulate the invasiveness of some particular, but not all S. Typhimurium strains.