Human parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen, failed to bind B19, whereas some cell lines had an ability to bind B19 in spite of undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the Ku80 gene enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19-binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with Glycophorin A or CD36, B cells with CD20 or T cells with CD3 were all positive for cell surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathological immunity associated with B19 infection.