The fibronectin-attachment protein (FAP) is conserved among several species of mycobacteria. Although this protein is associated with attachment and internalization of bacteria to host cells via fibronectin, the physiological role of the protein still remains unclear. To investigate this point, we generated FAP gene disruptant in Mycobacterium smegmatis. The gene disruption, verified by Southern blot and PCR analysis, induced changes on the bacteria, which are associated with strong aggregation and alteration of cell surface properties. Increased hydrophobicity and Congo red accumulation was observed in the FAP gene disruptant. In addition, the complementation experiment demonstrated that the corresponding gene restored wild type morphology in the disruptant. These results indicate that the FAP affects the cell surface properties, and its deletion lead to enhanced aggregation of the M. smegmatis.