Rotavirus is the most common cause of severe gastroenteritis in infants and children worldwide. The cell attachment of most animal rotaviruses, which belong to the neuraminidase-sensitive strains, requires sialic acid residues on the host cell membranes. On the other hand, most human rotaviruses are classified as neuraminidase-insensitive strains. The involvement of gangliosides on the host cell surface in human rotavirus infection was investigated by immunostaining analysis of target cells, and by assaying the neutralization of infection by rotavirus and the blocking of target cellular receptors. In host cells (MA104 cells) pretreated with Arthrobacter ureafaciens neuraminidase, which were still infected by human rotaviruses (KUN and MO strains), GM(3) was hydrolyzed markedly by the neuraminidase, while GM(1a) was not hydrolyzed at all. Infection by the rotaviruses was strongly inhibited by exogenous ganglioside GM(1a), but not GA(1). Infection was also inhibited by pretreatment of the MA104 cells with cholera toxin B-subunit, which specifically blocked ganglioside GM(1a) on the plasma membrane. The treatment of MA104 cells with the endoglycoceramidase attenuated human rotavirus infection. From these findings, we concluded that GM(1a) on the plasma membrane of the host cells was involved in the infection by human rotavirus KUN and MO strains.