We have previously shown that oral treatment with the leaf extract of the plant Kalanchoe pinnata (Kp) significantly decreases the lesion size and the parasite load in BALB/c mice infected with Leishmania amazonensis. Here we report on the mode of action of Kp, particularly on the induction of nitric oxide (NO) production by macrophages. We observed that Kp has no direct inhibitory activity on extracellular promastigotes, but effectively decreases the intracellular amastigote growth in a dose-related fashion. A 58% reduction in amastigote growth induced by 500 μg/ml Kp was associated with a 6-fold increase in the production of NO by the macrophages. IFN-γ synergistically enhanced the NO-stimulating effect of Kp in culture. Co-treatment with the inducible NO synthase enzyme inhibitor l-NG-monomethyl-arginine abolished the antileishmanial effect of Kp in vitro and in L. amazonensis-infected BALB/c mice. These results indicate that the protective effect of Kp in leishmaniasis may not be due to a direct effect on the parasite itself but rather to activation of the reactive nitrogen intermediates pathway of macrophages.