Updated publication reference for PubMed record(s): 34145271. An essential step in active host cell invasion by the obligate intracellular apicomplexan parasites is the formation of a moving junction (MJ), which joins both plasma membranes and connects the underlying host cortical cytoskeleton with the parasite actomyosin system. Invading Toxoplasma gondii secrete RON complex proteins from rhoptry organelles into the host cell which provide the MJ components on the cytoplasmic side of the host cell membrane. We investigated the role of an essential organelle-resident RON13 kinase and implicated in phosphorylation of rhoptry content. Structural biology and biochemistry demonstrated that RON13 is an atypical kinase inserted N-terminally in the organelle membrane and processed by the aspartyl protease 3. In the absence of RON13 function rhoptry discharge is unaffected but parasites fail to invade cells in vitro and become avirulent in a mouse model. Comparative phosphoproteomics revealed RON13-dependent phosphorylation of secreted RONs involved in anchoring the MJ in the host cell cytoskeleton.