Rotavirus infection leads to the death of half a million children annually. The exact specifics of interaction between rotavirus particles and host cells enabling invasion and infection have remained elusive. Host cell oligosaccharides are critical components, and their involvement aids the virus in cell-recognition and attachment, as well as dictation of the remarkable host-specificity that rotaviruses demonstrate. Interaction between the rotavirus spike-protein carbohydrate-binding domain (VP8*) and cell surface oligosaccharides facilitate virus recognition of host cells and attachment. Rotaviruses are considered, controversially, to recognise vastly different carbohydrate structures and either with incorporation of terminal sialic acid or without, as assessed by their ability to infect cells that have been pre-treated with sialidases. Herein, the X-ray crystallographic structures of VP8* from the sialidase insensitive Wa and the sialidase sensitive CRW-8 rotavirus strains that cause debilitating gastroenteritis in human and pig are reported. Striking differences are apparent regarding recognition of the sialic acid derivative methyl α-d-N-acetylneuraminide, presenting the first experimental evidence of the inability of the human rotavirus strain to bind this monosaccharide, that correlates with Wa and CRW-8 recognising sialidase-resistant and sialidase-sensitive receptors, respectively. Identified are structural features that provide insight in attainment of substrate specificity exhibited by porcine strains as compared to rhesus rotavirus. Revealed in the CRW-8 VP8* structure is an additional bound ligand that intriguingly, is within a cleft located equivalent to the carbohydrate-binding region of galectins, and is suggestive of a new region for interaction with cell-surface carbohydrates. This novel result and detailed comparison of our representative sialidase-sensitive CRW-8 and insensitive Wa VP8* structures with those reported leads to our hypothesis that this groove is used for binding carbohydrates, and that for the human strains, as for other sialidase-insensitive strains could represent a major oligosaccharide-binding region. Crown Copyright © 2007.