Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.