Different rates of bacterial translocation across the gut mucosa have been reported but few studies have examined translocation of commensals at the level of the gut epithelial microfold (M) cell. We used an in vitro M-cell model to quantify translocation and determine the transcriptional response of M cells to various commensal bacteria. The transport kinetics and gene expression profile of M cells in response to different bacterial strains, namely Lactobacillus salivarius, Escherichia coli and Bacteroides fragilis, was assessed. Bacterial strains translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency whereas L. salivarius translocated with less efficiency. Microarray analysis of the M cell response showed both common and differential gene expression changes between the bacterial strains. In the presence of bacteria, but not control beads, up-regulated genes were mainly involved in transcription regulation whereas pro-inflammatory and stress response genes were primarily up-regulated by E. coli and B. fragilis, but not L. salivarius nor beads. Translocation of bacteria and M-cell gene expression responses were confirmed in murine M cells following bacterial challenge in vivo. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.