Cyanovirin-N (CV-N) is a two-domain, cyanobacterial protein that inhibits human immunodeficiency virus (HIV) at nanomolar concentrations by binding to high mannose sugars on the HIV envelope glycoprotein gp120. The wild type protein can exist as a monomer or a domain-swapped dimer with the monomer and dimer containing two or four sugar binding sites, respectively, one on each domain. Here we demonstrate that monomeric, single binding site mutants are completely inactive and that a single site, whether located on domain A or B, is insufficient to impart the antiviral activity. Linking inactive, monomeric proteins in a head-to-head fashion by an intermolecular disulfide bond or by creating an exclusively domain-swapped dimer via a hinge residue deletion restored antiviral activity to levels similar to that of wild type CV-N. These findings demonstrate unequivocally that multisite binding by CV-N type lectins is necessary for viral inhibition.