Background. Fluoroquinolone (FQ) resistance is increasing worldwide among Salmonella spp. Among the mechanisms involved, increased efflux via the tri-partite AcrAB-TolC efflux system is mainly modulated through control of expression via the ramRA regulatory locus gene products. Interestingly, in some reference strains these have also been experimentally shown to regulate cell invasion-related genes of the type III secretion system-1 (T3SS-1). In this study we investigated whether natural mutations occurring in this locus in FQ-resistant S. enterica serovar Typhimurium (S. Typhimurium) epidemic clones resulted in the same effects. Methods. qRT-PCR and cell invasion assays were used to study three clinical FQ-resistant S. Typhimurium isolates representative of the DT104 and DT204 epidemic clones. For comparison three control reference quinolone-susceptible strains were included. Results. As previously shown, the investigated mutations altering RamR 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 the FQ-resistant isolate. Indeed, a ramA-dependent decreased invasion of intestinal epithelial cells was only observed for a particular clinical ramR mutant. Conclusion. ramRA mutations occurring in clinical FQ-resistant S. Typhimurium isolates may negatively modulate their invasiveness but this is strain-dependent.