Interaction of pathogenic bacteria with human cells is usually an essential step in the infection process. The bacterial invasion is stimulated by microbial binding to mammalian extracellular matrix proteins such as vitronectin, fibronectin or integrins. We have recently shown that some strains isolated from a clinical environment are able to grow at/or above 37°C. In particular, we demonstrated that P. fluorescens AF181 binds specifically to the surface of A549 human respiratory epithelial cells and that adhesiveness modulates the inflammatory response. In this study, the involvement of Alpha(v)Beta5 integrins and its respective natural ligand vitronectin (VN) in P. fluorescens AF181 adherence and invasion was examined. The host cell cytoskeleton and cellular tyrosine kinases seem to be solicited during the P. fluorescens-respiratory cell interaction; consequently, cytochalasin D and genistein decreased the bacterial adherence and internalization. Gene silencing of αv, β5 integrins and vitronectin reduced P. fluorescens adherence and internalization to A549 cells. Taken together, these findings suggest that Alpha_vBeta₅ integrins and their natural ligand VN are involved in P. fluorescens adherence and invasion in human epithelial cells.