To evaluate the role of V3-specific IgG antibodies (Abs) in the RV144 clinical HIV vaccine trial, which reduced HIV-1 infection by 31.2%, the anti-V3 Ab response was assessed. Vaccinees' V3 Abs were highly cross-reactive with cyclic V3 peptides (cV3s) from diverse virus subtypes. Sieve analysis of CRF01_AE breakthrough viruses from 43 vaccine- and 66 placebo-recipients demonstrated an estimated vaccine efficacy of 85% against viruses with amino acids mismatching the vaccine at V3 site 317 (p=0.004) and 52% against viruses matching the vaccine at V3 site 307 (p=0.004). This analysis was supported by data showing vaccinees' plasma Abs were less reactive with I ³⁰⁷ replaced with residues found more often in vaccinees' breakthrough viruses. Simultaneously, viruses with mutations at F ³¹⁷ were less infectious, possibly due to the contribution of F ³¹⁷ to optimal formation of the V3 hydrophobic core. These data suggest that RV144-induced V3-specific Abs imposed immune pressure on infecting viruses and inform efforts to design an HIV vaccine.