Background The treatment for leishmaniasis is currently based on pentavalent antimonials and amphotericin B; however, these drugs result in numerous adverse side effects. The lack of affordable therapy has necessitated the urgent development of new drugs that are efficacious, safe, and more accessible to patients. Natural products are a major source for the discovery of new and selective molecules for neglected diseases. In this paper, we evaluated the effect of apigenin on Leishmania amazonensis in vitro and in vivo and described the mechanism of action against intracellular amastigotes of L. amazonensis. Methodology/Principal Finding Apigenin reduced the infection index in a dose-dependent manner, with IC50 values of 4.3 μM and a selectivity index of 18.2. Apigenin induced ROS production in the L. amazonensis- infected macrophage, and the effects were reversed by NAC and GSH. Additionally, apigenin induced an increase in the number of macrophages autophagosomes after the infection, surrounding the parasitophorous vacuole, suggestive of the involvement of host autophagy probably due to ROS generation induced by apigenin. Furthermore, apigenin treatment was also effective in vivo, demonstrating oral bioavailability and reduced parasitic loads without altering serological toxicity markers. Conclusions/Significance In conclusion, our study suggests that apigenin exhibits leishmanicidal effects against L. amazonensis-infected macrophages. ROS production, as part of the mechanism of action, could occur through the increase in host autophagy and thereby promoting parasite death. Furthermore, our data suggest that apigenin is effective in the treatment of L. amazonensis-infected BALB/c mice by oral administration, without altering serological toxicity markers. The selective in vitro activity of apigenin, together with excellent theoretical predictions of oral availability, clear decreases in parasite load and lesion size, and no observed compromises to the overall health of the infected mice encourage us to supports further studies of apigenin as a candidate for the chemotherapeutic treatment of leishmaniasis.