Cyanovirin-N (CVN) is a novel cyanobacterial protein that potently inhibits viral entry by human immunodeficiency viruses (HIV) via high affinity carbohydrate-mediated binding to the surface envelope glycoprotein gp120. Bearing C(2) pseudo-symmetry, CVN contains two carbohydrate binding sites of differing affinities located at opposite ends of the protein. CVN selectively binds with nanomolar affinity the mammalian high mannose oligosaccharides oligomannose-8 D1D3 and oligomannose-9, which also govern binding to gp120. At nanomolar concentrations CVN binds these oligosaccharides only through its high affinity site, while at micromolar to millimolar concentrations the oligosaccharides are bound through both sites leading to divalent protein-carbohydrate interactions. Similarly, two modes of binding to gp120 can be envisioned where CVN either binds gp120 solely through the high affinity site, or binds divalently using both carbohydrate binding sites. To determine the role of the low affinity site in binding to gp120, we sought to design a variant of CVN that lacks the low affinity carbohydrate binding site but retains a fully functional high affinity site. Thus, we constructed a series of CVN mutants possessing cumulative mutations in the low affinity site only, and characterized by NMR the overall structure and carbohydrate binding ability of each of these mutants. We demonstrate that carbohydrate binding by the low affinity site is completely absent in two mutants bearing three or four mutations (namely, m3-CVN=Lys3Asn, Glu23Ile, Asn93Ala; and m4-CVN=Lys3Asn, Thr7Ala, Glu23Ile, Asn93Ala), while the high affinity site binds the high affinity ligand Manalpha(1-2)Manalpha with a K(d) value equal to that measured for CVN. Using an HIV-1 cell fusion assay, we show that all of the mutants inhibit HIV-1 fusion with nearly identical IC50 values as wild-type CVN. We interpret these results as indicating that the low affinity carbohydrate binding site of CVN is not necessary for high affinity binding to gp120, and HIV-1 fusion can therefore be blocked by monovalent protein-carbohydrate interactions.