Noroviruses are important agents of human gastroenteritis characterized by extensive sequence variation in the major capsid structural protein that likely encodes critical antigenic determinants of protective immunity. The lack of an infection model has limited detailed characterizations of viral antigenic relationships and identification of the essential components for protective immunity. This information would contribute to efficacious vaccine design against a broad array of norovirus strains. To understand the extent of heterotypic norovirus antibody specificity to inter- and intra-genogroup strains and its applicability to vaccine design, we collected sera from humans infected with different norovirus strains and from mice inoculated with alphavirus vectors expressing strain-specific recombinant norovirus-like particles (VLPs). We used VLPs that were assembled from Norwalk virus (NV), Hawaii virus (HV), Snow Mountain virus (SM) and Lordsdale virus (LV) as antigens to define and compare heterotypic antibody responses in humans and mice. We also examined if these heterotypic antibodies could block specific binding of ABH histo-blood group antigens, putative receptors for norovirus binding and entry, to norovirus VLPs. Furthermore, we examined the effect of multivalent inocula on the specificity, titer, and ligand blockade properties of systemic and mucosal norovirus-specific antibodies in mice. Our studies suggest that infection with one of several different genogroup I (GI) strains in humans induces heterotypic antibodies that block NV binding to ABH antigens, although comparable findings were not evident following infection with genogroup (GII) strains. Additionally, inoculating mice with vaccine cocktails encoding multiple norovirus VLPs enhances heterotypic and ligand attachment-blocking antibody responses against the LV strain not included in the cocktail. These data suggest that multivalent vaccination may provide better protection from a broader range of noroviruses than monovalent vaccination.