The membrane protein NINJ1 plays a crucial role in plasma membrane rupture during pyroptosis and other lytic cell death pathways. This study investigates the mechanism by which NINJ1 mediates this process. Using cryo-EM, the structure of a NINJ1 oligomer was determined, revealing a unique conformation with a concave hydrophobic side and a convex hydrophilic side. NINJ1 oligomers form rings that can encircle and release membrane disks, leading to membrane fragmentation and rupture. Live cell imaging and super-resolution microscopy confirmed the release of NINJ1-encircled membrane disks into the culture supernatant. Mutational analyses identified key residues involved in NINJ1 oligomerization and function. The findings suggest that NINJ1-mediated membrane lysis involves a "cookie cutter" mechanism, where NINJ1 rings cut and release membrane patches, rather than a pore formation mechanism. This study provides new insights into the molecular mechanisms underlying pyroptosis and other lytic cell death pathways.The membrane protein NINJ1 plays a crucial role in plasma membrane rupture during pyroptosis and other lytic cell death pathways. This study investigates the mechanism by which NINJ1 mediates this process. Using cryo-EM, the structure of a NINJ1 oligomer was determined, revealing a unique conformation with a concave hydrophobic side and a convex hydrophilic side. NINJ1 oligomers form rings that can encircle and release membrane disks, leading to membrane fragmentation and rupture. Live cell imaging and super-resolution microscopy confirmed the release of NINJ1-encircled membrane disks into the culture supernatant. Mutational analyses identified key residues involved in NINJ1 oligomerization and function. The findings suggest that NINJ1-mediated membrane lysis involves a "cookie cutter" mechanism, where NINJ1 rings cut and release membrane patches, rather than a pore formation mechanism. This study provides new insights into the molecular mechanisms underlying pyroptosis and other lytic cell death pathways.