The Caliciviridae include many notable human and animal pathogens, including norovirus and sapovirus which cause outbreaks of acute gastroenteritis. Feline Calicivirus (FCV) is a member of the Caliciviridae that causes acute upper respiratory infections in cats, and is commonly used as a cultivable surrogate for human norovirus. Vaccine-resistant virulent systemic (VS) strains of FCV have recently emerged, which are associated with multi-organ failure and high morbidity and mortality. We previously demonstrated that following receptor engagement, FCV assembles a portal structure at a unique capsid three-fold axis. This comprises twelve copies of the minor capsid protein VP2 and is essential for genome delivery. We designed a short peptide based on structural data that occludes the VP2 binding sites on the capsid surface, to prevent assembly of the VP2 portal and thereby halt the viral entry mechanism. Incubation with low micromolar concentrations of the peptide considerably reduced the infectivity of several clinical isolates of FCV associated with virulent systemic disease. Cryo-electron microscopy structures of FCV virions complexed with the peptide confirmed that it occupies the VP2 binding site on the major capsid protein VP1, preventing portal assembly and stopping genome delivery. Therefore, our data show that targeting the VP2 protein, which is highly conserved across the Caliciviridae, is a viable antiviral approach to preventing calicivirus infection.