The incidence of multi-drug resistant Salmonella has increased globally over the past several decades and has become a major public health concern. Isolates of Salmonella enterica serovar Typhimurium DT104 are resistant to five or more antibiotics, including florfenicol, and have been associated with enhanced virulence in livestock and humans. Because sub-inhibitory concentrations of some antibiotics have been found to modulate invasion of certain bacteria under specific conditions, the effect of florfenicol on S. Typhimurium DT104 invasion was evaluated. Three clinical bovine isolates were exposed to sub-inhibitory concentrations of florfenicol for 30 minutes to establish the initial response to the antibiotic. HEp-2 cellular invasion assays, as well as expression analyses of invasion-related genes, demonstrated that the invasiveness of the S. Typhimurium DT104 isolates was not enhanced after exposure to sub-inhibitory concentrations of florfenicol. These results suggest that cattle and swine can be treated with florfenicol for respiratory illness without exacerbating Salmonella Typhimurium DT104 virulence in carrier animals.