Toxoplasma gondii is an intracellular parasite that can infect many host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection. The vast majority of these secreted proteins have no predicted functional domains and remain uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, is essential for virulence and has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. Finally, we show that ROP1 co-immunoprecipitates with the host cell protein C1QBP, an emerging regulator of innate immune signaling. In summary, we identify putative in vivo virulence factors in the T. gondii Prugniaud strain and show that ROP1 is an important and previously overlooked effector protein that counteracts both murine and human innate immunity. Author summary: Toxoplasma gondii is a single-celled eukaryotic pathogen that can infect many different species, including mice and humans. T. gondii secretes a large number of proteins into host cells that it infects, although the majority of these proteins are not well studied. We have carried out a knockout screen to identify T. gondii genes that are important for the parasite to survive during infection of a mouse. One of the genes we identified encodes the parasite protein ROP1, which was shown 30 years ago to be secreted into the host cell, but whose function remains unknown. We show that deletion of ROP1 causes an otherwise lethal infection to be efficiently cleared by the host immune system. ROP1 is important for T. gondii to evade the cell autonomous immune responses of both human and murine cells, which is usual as the key mechanisms that control intracellular pathogens differ between humans and mice. ROP1 may interact with the host protein C1QBP, indicating the direction of future work to establish a mechanistic link to immune evasion. [ABSTRACT FROM AUTHOR]