Current study aims to characterize the influence of sub-minimum inhibitory concentration (sub-MIC) of ciprofloxacin on Salmonella intracellular survival and host virulence. Herein, Salmonella resistance patterns to various antibiotics were in agreement with those reported in previous studies. Moreover, intracellular survival of both ciprofloxacin-sensitive and -resistant Salmonella was markedly reduced upon treatment with sub-MIC of ciprofloxacin as determined by gentamicin protection assay. These findings were further confirmed using immunostaining indicating an inhibitory effect of sub-MIC of ciprofloxacin on Salmonella intracellular survival. RT-qPCR revealed that expression of genes encoding Salmonella type three secretion system (TTSS) decreased upon bacterial exposure to sub-MIC of ciprofloxacin. Furthermore, bacterial exposure to sub-MIC of ciprofloxacin significantly reduced expression of both sifA and sifB, which are important for Salmonella filaments formation within the host. Treatment of Salmonella with sub-MIC of ciprofloxacin reduced bacterial capacity to kill mice infection models. A lower mortality rate was observed in mice injected with Salmonella treated with sub-MIC of ciprofloxacin as compared with mice inoculated with untreated bacteria. Collectively, current findings indicate that, in addition to its bactericidal potential, sub-MIC of ciprofloxacin could inhibit Salmonella intracellular survival, virulence genes expression as well as host pathogenesis, providing another mechanism for ciprofloxacin in limiting Salmonella host infection.