Streptococcus uberis, an environmental mastitis pathogen, is an important causative agent of mastitis in dairy cattle throughout the world. Research from our laboratory demonstrated that bovine lactoferrin (LF), a whey protein present in milk and nonlactating cow mammary secretions, significantly enhanced adherence of S. uberis to mammary epithelial cells in culture. Subsequent research from our laboratory identified S. uberis adhesion molecule (SUAM) showing an affinity for LF. The objective of the present investigation was to test the hypothesis that the interaction between SUAM, bovine LF, and a putative LF receptor on the bovine mammary epithelial cell surface could serve as a bridging molecule for internalization of S. uberis into mammary epithelial cells. When internalization assays were conducted using cell growth medium containing bovine LF, a significant increase in internalization of S. uberis into mammary epithelial cells was observed. However, this effect was reversed when assays were conducted in the presence of antibodies to bovine LF suggesting that internalization of S. uberis into mammary epithelial cells, at least in part, was mediated by LF ligands. When S. uberis was pretreated with antibodies to SUAM, internalization in the presence of LF was reduced in the same manner as observed with antibodies to LF. Transmission and scanning electron microscopy results demonstrated that streptavidin-coated gold particles specifically localized on biotinylated LF receptors on S. uberis and mammary epithelial cell surfaces supporting the availability of LF receptors. Collectively, these results suggest that LF serves as a bridging molecule between SUAM located on the surface of S. uberis and LF receptors located on the surface of mammary epithelial cells thus enhancing internalization of S. uberis into host cells. Exploitation of LF as a molecular bridge for internalization of S. uberis into mammary epithelial cells may confer a significant advantage allowing mammary gland infection.