By immunizing mice sequentially with six different V3 peptides we obtained a murine monoclonal antibody (MAb) C25, and its humanized counterpart KD-247. The MAb recognizes the sequence IGPGRA at the tip of the V3 loop and displays broad neutralizing activity against a variety of HIV-1 isolates. KD-247 was tested in an ex vivo neutralization assay to determine its capability to contain the spread of a quasi species population of clade B HIV-1 derived from two patients. The epitope of KD-247 was generally matching with the V3 sequences of various clones isolated from plasma and peripheral blood mononuclear cells (PBMC) of two patients. Complete or strong inhibition of viral replication was observed when the patients' PBMC were cultured with a high concentration of KD-247. Some neutralization escape variants, which had mutations in the V3 or outside of the V3 loop, emerged only at a low concentration of the MAb. These results suggest that KD-247 could be a good candidate for immunotherapy against HIV-1 in vivo.