Two LipL32-specific mouse monoclonal antibodies (mAbLPF1 and mAbLPF2) which neutralized Leptospira-mediated hemolysis in vitro and rescued hamsters from lethal Leptospira infection were produced. In this communication, locations and characteristics of the protective epitopes of the mAbs were studied by using a truncated LipL32 recombinant protein based-immunoassay and phage consensus mimotope identification and multiple alignments. The mAbLPF1 epitope consisted of P243, L244, I245, H246, L252 and Q253 on the LipL32 protein; it is mapped on the surface-exposed region of non-continuous β13-turn and C-terminal amphipathic α6 helix with hydrophobic patch, contributing to phospholipid/host cell adhesion and membrane insertion on one side, and hydrophilic, acidic and basic amino acid residues on another side. The epitope peptide of the mAbLPF2 is linear 122PEEKSMPHW130 and located on surface-exposed α1 and α2 between β7 and β8 that bound to several host constituents. Both epitopes are highly conserved among the pathogenic and intermediately pathogenic Leptospira spp. and are absent from the LipL32 superfamily proteins of other microorganisms. This study not only enlightens the molecular mechanisms of the therapeutic mAbLPF1 and mAbLPF2, but also elaborates the potential of the two LipL32 regions as diagnostic and vaccine targets for leptospirosis.