Bacteroides fragilis is the Gram-negative strict anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteremia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them were already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules along the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized in latex beads it was observed an increased adhesion on the mutant strains when compared to the wild type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer membrane protein and an alteration on the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.