An HIV-1-specific miniantibody, a peptide representing the third heavy chain complementarity-determining region (CDR) of an HIV-specific mouse antibody, was characterized and modified with unnatural D-isomeric amino acids. The CDR peptide and its parent antibody bound to a similar epitope, located in the V3 region of HIV-1 gp120. A shortened CDR sequence was modified with D-amino acids to create an all-D-amino acid retro-inverso (RI) peptide with a reversed sequence order. The RI CDR was less susceptible to proteolytic degradation than its L-counterpart and had a higher affinity for HIV-1 peptides. The miniantibody and its parent antibody showed neutralization of both primary and laboratory strains of HIV-1. In accordance with the binding studies, the RI CDR showed a stronger HIV-inhibiting capacity than its L-counterpart. We conclude that the anti-HIV retro-inverso CDR identified in this study has the potential to become a future anti-HIV drug. It has a virus-neutralizing capacity in vitro and appears to be stable. Future research should focus on characterizing its antiviral activity in vivo.