Leishmaniasis is caused by species of the protozoan parasite Leishmania. It is the third most important vector-borne disease and is widely distributed throughout the world. The World Health Organization recommends pentavalent antimonials as drugs of first choice in its treatment. Although Glucantime has traditionally been used to treat leishmaniasis, there are still many questions about its structure, mechanisms of action and ability to induce damage in DNA. In this study, the genotoxic activity of this drug was evaluated in vitro using human lymphocytes treated for 3 and 24 h (comet assay) and 48 h (apoptosis assay) with 3.25, 7.5 and 15 mg/ml of Glucantime, respectively, corresponding to 1.06, 2.12 and 4.25 mg/ml of pentavalent antimony. In the in vivo tests, Swiss mice received acute treatment with three doses (212.5, 425 and 850 mg/kg) of pentavalent antimony. All the treatments were administered intraperitoneally in the volumes of 0.1 ml/10 g of body weight, adapting human exposure to murine conditions. The animals were treated for 3 h in the comet assay using resident peritoneal exudate macrophages, for 24 h in the comet assay using peripheral blood leukocytes and for 24 h in the bone marrow erythrocyte micronucleus test. While no genotoxic effect was observed in the in vitro tests, the in vivo tests showed that Glucantime induces DNA damage. These findings indicate that Glucantime is a promutagenic compound that causes damage to DNA after reduction of pentavalent antimony (SbV) into the more toxic trivalent antimony (SbIII) in the antimonial drug meglumine antimoniate.