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The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl α-D-N-acetylneuraminide (Neu5Acα2Me) and the disaccharide methyl S-(α-D-N-acetylneuraminosyl)-(2→6)-6-thio-β-D-galactopyranoside (Neu5Ac-α(2,6)-S-Galβ1Me). In the VP8*–disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*–disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-α-D-N-acetylneuraminide (Neu5,9Ac2α2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide''s methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein–Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acα2Me would not be sustained. Neu5Acα2Me, Neu5Ac-α(2,6)-S-Galβ1Me, Neu5,9Ac2α2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection. [ABSTRACT FROM AUTHOR]