Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.