We tested the hypothesis that cathepsins and specifically toxopain-1, a cathepsin B, play a critical role in the pathogenesis of toxoplasmosis. We found that inhibiting the expression of toxopain-1-specific mRNA and protein by >60% significantly decreased the capacity of the parasites to multiply and invade in vitro. To relate these in vitro results to the role of toxopain-1 in pathogenesis in vivo, we developed a novel chicken embryo model of congenital toxoplasmosis. Inhibiting either toxopain-1 expression or specific cysteine proteinase activity significantly reduced congenital infection of chicken embryos, as determined by histopathology and by the number of parasites quantified by real-time PCR. Our new model provides key in vivo validation for the hypothesis that toxopain-1 is a potential drug target in Toxoplasma gondii and also provides a new animal model for rapid, inexpensive screening of antiparasitic compounds.