Egress from the host cell is a crucial step in the infectious cycle of intracellular pathogens. Intracellular pathogens have thus evolved a variety of mechanisms that drive successful exit from host cells. Apicomplexan parasites employ pore-forming proteins that disrupt cellular membranes, including members of the membrane attack complex and perforin (MACPF) family, to either traverse cells during tissue migration or egress from the parasitophorous vacuole (PV) after asexual replication. Apicomplexan MACPF proteins contain a conserved pore-forming MACPF domain and a membrane binding apicomplexan-specific C-terminal domain that contains a b-rich repeat and basic C-terminal tail. T. gondii Perforin-like protein 1 (TgPLP1) is necessary for efficient tachyzoite egress, and the C-terminal domain is required for cytolytic activity. The structural basis for membrane binding and the molecular determinants for membrane recognition, however, remain unknown. To address this we solved the 1.13 Å resolution crystal structure of the TgPLP1 b-rich domain. The structure reveals an unusual double-layered b-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the b-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence indicate insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we generated a series of mutant strains harboring amino acid deletions and substitutions in the hydrophobic loop and assessed their egress competence. Strikingly, virtually all of the mutant parasite strains have an egress deficiency that is equal to that of TgPLP1 knockout parasites, including alanine substitution of the aforementioned tryptophan. Together our findings suggest a crucial role for the hydrophobic loop in rendering a membrane anchor for TgPLP1 to support its cytolytic activity and function in egress.