Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identified and characterized an important chaperone-like GRA in Toxoplasma and provided a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ- activated macrophages.