Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa. To facilitate high-efficiency invasion of host cells, T. gondii secretes various proteins related to the moving junction (MJ) complex from rhoptries and micronemes into the interface between the parasite and host. AMA1/RON2/4/5/8 is an important MJ complex, but its mechanism of assembly remains unclear. In this study, we used the CRISPR-Cas9 system to generate a derivative of T. gondii strain RH with a null mutation in TgRON4, thought to be an essential MJ component. Deficiency of TgRON4 moderately decreased invasion ability relative to that of the wild-type parasite. In addition, expression of the endogenous N-terminal fragment of RON5 decreased in the mutant. Together, the results improve our understanding of the assembly mechanism of the MJ complex of T. gondii and raise the possibility of developing new therapeutic drugs that target this complex.