Hydrophilic albumin microspheres are proposed as a new delivery system for amphotericin B (AMB; AMB microspheres). The acute toxicity of AMB microspheres was lower than that of the AMB-deoxycholate (AMB-Doc) reference formulation in hamsters. Lethal doses in healthy and infected animals were improved at least eight times. Intravenous bolus administration of doses of AMB microspheres up to 40 mg/kg of body weight did not produce acute symptoms of toxicity. The efficacy of this new formulation was tested against Leishmania infantum-infected hamsters at doses of 2, 10, 20, and 40 mg/kg. With the 2-mg/kg dose, the activity of AMB, as assessed through the parasite load reductions in the liver and spleen and the evolution of antibody levels, was also improved (P < 0.05) by use of the AMB microsphere system. At the higher doses of 10, 20, and 40 mg/kg, reductions in parasite levels of more than 99% were achieved in the liver and spleen after the administration of AMB microspheres. A pharmacokinetic study was performed to study the serum, liver, and spleen AMB concentrations after administration of AMB microspheres and the reference formulation. Interestingly, a significant accumulation of AMB in the spleen and liver was observed after AMB microsphere administration. Our results suggest that this new formulation is a promising alternative to the conventional AMB-Doc formulation for the treatment of visceral leishmaniasis.