Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. kappa-Casein (kappa-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine kappa-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, kappa-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in kappa-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95 degrees C for 30 min) in a viral titer-dependent manner. Although the heated kappa-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine kappa-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in kappa-CN may play an important role in maintenance of kappa-CN binding to HRV.