Due to the different mechanisms HIV-1 has evolved to escape from a neutralizing antibody response it has been extremely challenging to develop an effective anti-HIV-1 vaccine. The V3 region of the gp120 HIV-1 envelope glycoprotein has been considered as one of the possible targets for an anti-HIV vaccine. It is well known that the V3 region of gp120 is at least partially masked in circulating strains and becomes exposed only after CD4 binding. However, when the virus is bound to surface CD4, steric hindrance prevents effective neutralization by V3-directed antibodies. Here we have used a 27-residue CD4-mimetic peptide in combination with immune sera elicited by an optimally constrained V3 peptide to enhance neutralization of a panel of clade B viruses. We observed strong synergism between the immune sera and the CD4-mimetic in the neutralization of tier 1 and a representative tier 2 clade B virus suggesting that the constrained V3 peptide immunogen correctly mimics the V3 conformation even in tier 2 clade B viruses. This synergy should improve the potential of CD4-mimetic compounds for preexposure prophylaxis and in the treatment of HIV-1-infected patients who usually manifest high titers of V3-directed antibodies. Moreover, constrained V3 immunogens elicit immune sera that may neutralize HIV in synergy with CD4 binding site antibodies that expose V3 and the coreceptor binding site.