is the causing agent of visceral leishmaniasis, a common infection that affects millions of people from the most underdeveloped countries. Miltefosine is the only oral drug to treat infections caused by Nevertheless, its mechanism of action is not well understood. While miltefosine inhibits the synthesis of phosphatidylcholine and also affects the parasite mitochondrion, inhibiting the cytochrome oxidase, it is to be expected that this potent drug also produces its effect through other targets. In this context, it has been reported that the disruption of the intracellular Ca homeostasis represents an important object for the action of drugs in trypanosomatids. Recently, we have described a plasma membrane Ca channel in , which is similar to the L-type voltage-gated Ca channel (VGCC) present in humans. Remarkably, the parasite Ca channel is activated by sphingosine, while the L-type VGCC is not affected by this sphingolipid. In the present work we demonstrated that, similarly to sphingosine, miltefosine is able to activate the plasma membrane Ca channel from Interestingly, nifedipine, the classical antagonist of the human channel, was not able to fully block the parasite plasma membrane Ca channel, indicating that the mechanism of interaction is not identical to that of sphingosine. In this work we also show that miltefosine is able to strongly affect the acidocalcisomes from , inducing the rapid alkalinization of these important organelles. In conclusion, we demonstrate two new mechanisms of action of miltefosine in , both related to disruption of parasite Ca homeostasis.