Generation of abasic (AP) sites is one of the main anomalies to arise in cellular DNA. These lesions are highly mutagenic, and need to be repaired by the base-excision repair (BER) system. Oxidative stress and misincorporation of dUTP are important sources of mutation load trough generation of AP sites. Kinetoplastid protozoa are able to survive in a highly oxidative environment within the host macrophages and between the different strategies used for survival, active DNA repair mechanisms must exist. In order to assess the role of BER in protecting parasites against DNA damage, we have overexpressed one enzyme of the pathway, AP endonuclease, in Leishmania major. Parasites overproducing AP endonuclease of L. major (APLM) showed an increased resistance to hydrogen peroxide, a mutagen that produces oxidative stress, and also to methotrexate (MTX), an inhibitor of thymidylate biosynthesis which causes a massive incorporation of dUTP into DNA, when compared to control cells. Moreover, DNA fragmentation caused by MTX was prevented in cells overexpressing APLM. Our results suggest that APLM is a key enzyme in mediating repair of AP sites in these pathogens.