Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324-684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.