Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44_720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44_720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44_720-1138 but not in HGP44_720-1081, could bind HGP44_720-1081 in a dose-dependent manner and effectively inhibited HGP44_720-1081-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44_720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44_720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44_720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 × 10⁻⁷ M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.