Blastocystis is an enteric protistan parasite of zoonotic potential and poorly understood pathogenesis. We have previously reported that Blastocystis cysteine proteases can degrade human secretory IgA and are also responsible for the induction of IL-8 response in colonic epithelial cells in vitro. Differences in virulence between Blastocystis subtypes have been reported recently in both animal models and clinical studies, although cellular mechanisms for these differences are currently unknown. Parasites such as Giardia intestinalis and Entamoeba histolytica have distinct virulent and non-virulent strains which may be attributable to variations in their cysteine proteases. In the present study, variations in cysteine protease activity was observed between avian (subtype 7) and rodent (subtype 4) isolates of Blastocystis with avian isolates exhibiting approximately two times higher peak cysteine protease activity than rodent isolates. Cysteine protease activity and parasite cell size varied over time within cultures of the same isolate. An association between parasite cell size and protease activity was observed.