Aims: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin-binding protein. Methods and Results: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease-sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco-2 or HT-29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose-dependent manner. Western blot experiments with FbpA-specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole-cell ELISA. Conclusions: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. Significance and Impact of the Study: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.