Rotavirus infection causes diarrhea to humans, animals and birds. The NSP4 protein of Group A rotavirus has been recognized as a viral enterotoxin. This single protein plays important roles in viral pathogenesis and morphogenesis. Domains involved in structure and biologic functions have been proposed mainly based on the SA11 strain, a prototype of group A rotavirus. NSP4 has been classified into different genotypes based on sequence homology. These analyses are based on representative strains selected but not comprehensive. In this paper, we collected all NSP4 sequences in the GenBank and performed a detailed computational analysis. Our analysis of 176 NSP4 proteins in Groups A, B and C rotaviruses confirms that the recently published avian NSP4 sequences belong to a new genotype (Mori Y., Borgan M.A., Ito N., Sugiyama M. and Minamoto N., Virus Res 89, 145–151, 2002), besides the four known NSP4 genotypes of Group A mammalian rotaviruses. Significant differences were discovered in the physicochemical properties between the avian and mammalian NSP4 proteins. In particular, lack of a highly probable coiled-coil region in the avian sequences implies a diversion of the NSP4 quaternary structure from the latter, although the secondary and tertiary structures may be similar. Fourteen amino acids are found absolutely conserved in the Group A NSP4 sequences, regardless of genotype. Of the conserved residues, two are glycosylation sites, one is in the middle of the transmembrane segment, seven span the VP4 binding domain, and five are clustered in the middle of the toxic peptide region, indicating the functional importance of the conservation.