Updated project metadata. Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations (large (LEV) and small extracellular vesicles (SEV)) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from Giardia and their putative effects on host-pathogen interactions. PAD-inhibitor Cl-amidine and CBD were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells in vitro. The strong efficacy of the PAD-inhibitor on Giardia EV release indicates a phylogenetically conserved pathway of PAD-mediated EV release, most likely affecting the Giardia arginine deiminase (GiADI) homolog of mammalian PADs. Our results suggest that LEVs and SEVs are differently involved in protozoa communication, and that treatment with EV-inhibitors may be a novel strategy for recurrent giardiasis treatment.