Hypothesis: Artemisinin, a potential antileishmanial compound with poor bioavailability and stability has limited efficacy in visceral leishmaniasis. Encapsulating artemisinin into poly lactic-co glycolic nanoparticles may improve its effectiveness and reduce toxicity. Experiments: Artemisinin-loaded nanoparticles were prepared, optimized (using Box-Behnken design) and characterized by dynamic light scattering technique, Atomic force microscopy (AFM), Transmission electron microscopy (TEM) and Fourier Transform-Infra Red spectroscopy. Release kinetics of artemisinin from optimized nanoformulation was studied by dialysis method at pH 7.4 and 5.5. Cytotoxicity and antileishmanial activity of these nanoparticles was tested on murine macrophages by MTT assay and macrophage-infested Leishmania donovani amastigotes ex vivo, respectively. Findings: Artemisinin-loaded nanoparticles were 221 +/- 14 nm in diameter, with polydispersity index, zeta potential, drug loading and entrapment efficiency of 0.1 +/- 0.015, -9.07 +/- 0.69 mV, 28.03 +/- 1.14 and 68.48 +/- 1.97, respectively. AFM and TEM studies indicated that the particles were spherical in shape. These colloidal particles showed a sustained release pattern in vitro. Treatment with artemisinin-loaded nanoparticles significantly reduced the number of amastigotes per macrophage and percent infected macrophages ex vivo compared to free artemisinin. These nanoparticles were also non-toxic to macrophages compared to artemisinin alone. (C) 2014 Elsevier Inc. All rights reserved.