Hantavirus-induced diseases such as hantavirus cardiopulmonary syndrome and hemorrhagic fever with renal syndrome are a global health concern. Hantavirus cardiopulmonary syndrome caused by Sin Nombre virus lacks specific therapy and its high mortality makes Sin Nombre virus a potential bioweapon agent. Sin Nombre virus entry into susceptible cells requires expression of αvβ3 integrin. We recently reported the sequence of a cyclic nonapeptide that inhibited Sin Nombre virus entry into Vero E6 cells at a level comparable to ReoPro, a Fab fragment of the anti-β3 antibody c7E3. Here, we refine the parental peptide, cyclo-[CPFVKTQLC], using alanine scanning and amino acid deletions, by optimizing for viral inhibition. The IC50 of the resulting peptide, cyclo-[CPFVC], was 267 μm compared with 263 μm for the parental peptide. The solution structure of cyclo-[CPFVC] was determined by two-dimensional nuclear magnetic resonance spectroscopy, revealing the Phe ring in an extended conformation stacked against the Pro ring and containing a β-turn encompassing Val-4 through Cys-1. As an initial step in identifying interactions between cyclo-[CPFVC] and its target cellular receptor, the refined peptide structure was docked into the ReoPro binding site of integrin β3. This structure will provide the basis for designing more potent peptidomimetic therapeutics to prevent Sin Nombre virus entry and treat hantavirus cardiopulmonary syndrome.