Human babesiosis is an emerging infectious disease caused by a blood-borne single-celled parasite belonging to the genus Babesia. Cases of human babesiosis are commonly reported in the United States, Western Europe, and Asia . In the United States, the two most common species are Babesia microti and Babesia duncani. Transmitted to humans through tick bites, the parasite infects the host’s red blood cells and induces flu-like symptoms, and has evolved mechanisms to manipulate the immune system, enabling its persistence. One key mechanism is the secretion of extracellular vesicles which carry bioactive molecules including proteins, lipids and genetic material that modulate pathogen-host interactions and disease development. The inhibition of the secretion of these vesicles may lead to disease control. One potential inhibitor of extracellular vesicle secretion is linoleic acid, a poly-unsaturated lipid that has demonstrated inhibitory properties in other parasites. To study the effects of development stage- dependent stimulus on Babesia duncani, we also examined the use of sorbitol, a sugar alcohol, to synchronize parasites. In this study, using a Babesia duncani in vitro continuous culture system and microscopy techniques, we showed sorbitol-induced synchronization of parasite development, and the inhibition of extracellular vesicle secretion.