Rotaviruses are important causes of infant morbidity and mortality worldwide. It has been previously shown that mucinous glycoproteins can inhibit rotavirus replication. However, the structure-function relationships of this inhibition have not been completely elucidated. Mucins were purified from epithelial scrapings of rat and human intestine by CsCl density-gradient ultracentrifugation and tested for the inhibition of rotavirus replication in MA-104 cells. Native human and rat intestinal mucins inhibited the replication of human and animal rotaviruses at low concentrations. Antiviral activity was most prominent in the densely glycosylated part of the rat and human mucins. Activity was retained after thiol reduction and alkylation, chloroform methanol partition, and partial removal of oligosaccharides. However, total deglycosylation of the mucins destroyed antiviral activity. Intestinal mucins from humans and other animals are potent inhibitors of rotavirus replication, and this inhibition is dependent on specific mucin-viral interactions.