Apicomplexa parasites, including Toxoplasma and Plasmodium species, possess a unique invasion mechanism that involves a tight apposition between the parasite and the host plasma membranes, called "moving junction" (MJ). The MJ is formed by the assembly of the microneme protein AMA1, exposed at the surface of the parasite, and the parasite rhoptry neck (RON) protein RON2, exposed at the surface of the host cell. In the host cell, RON2 is associated with three additional parasite RON proteins, RON4, RON5 and RON8. Here we describe RON4 L1 , an additional member of the MJ complex in Toxoplasma. RON4 L1 displays some sequence similarity with RON4 and is cleaved at the C-terminal end before reaching the rhoptry neck. Upon secretion during invasion, RON4 L1 is associated with MJ and targeted to the cytosolic face of the host membrane. We generated a RON4 L1 knock-out cell line and showed that it is not essential for the lytic cycle in vitro, although mutant parasites kill mice less efficiently. Similarly to RON8, RON4 L1 is a coccidian-specific protein and its traffic to the MJ is not affected in absence of RON2, RON4 and RON5, suggesting the co-existence of independent MJ complexes in tachyzoite of Toxoplasma.