Apicomplexan parasites are a leading cause of human and livestock diseases worldwide, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan, and outline several lessons that emerge from it. Using CRISPR/Cas9, we assess the contribution of each Toxoplasma gondii gene to parasite fitness during infection of human fibroblasts. This analysis defined ~200 fitness-conferring genes unique to the phylum, from which 16 previously uncharacterized proteins were further investigated. Analysis of their localization revealed a preponderance of these important hypothetical proteins localized to the mitochondrion. One of these proteins was predicted to have structural similarity to eukaryotic ATP synthase stator subunits, which have failed to be identified through sequence-based searches in apicomplexans. Through biochemical and genetic approaches, we have been able to confirm that this putative apicomplexan stator subunit indeed participates in the ATP synthase complex and is essential for its function. Additionally, secondary screens of the uncharacterized genes unique to apicomplexans identified the novel invasion factor claudin-like apicomplexan microneme protein (CLAMP), which displays similarity to mammalian tight-junction proteins and localizes to secretory organelles. CLAMP is found in all sequenced apicomplexan genomes, and its ortholog is essential during the asexual stages of the malaria parasite Plasmodium falciparum. Taken together, these results provide broad-based functional information on T. gondii genes and will facilitate future genetic approaches, expanding the horizon of antiparasitic interventions.