NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. The study reports the cryo-EM structure of a NINJ1 oligomer, revealing its role in membrane fragmentation. NINJ1 forms rings and filaments, and its oligomerization leads to membrane rupture by cutting and releasing membrane disks. The structure shows that NINJ1 has amphipathic and transmembrane helices, with kinked regions important for function. The oligomer has a concave hydrophobic side facing the membrane and a convex hydrophilic side formed by the amphipathic helices. NINJ1 can encircle and release membrane disks, consistent with membrane fragmentation. Live cell and super-resolution imaging show ring-like structures on the plasma membrane and released into the culture supernatant. Released NINJ1-encircled disks contain membrane, indicating NINJ1-mediated membrane rupture differs from gasdermin-mediated pore formation. NINJ1 forms rings and is released during pyroptosis, with live cell imaging revealing NINJ1 rings in the culture media. Mutational analyses confirm the importance of Gly residues at the TM kinks and positively charged residues for NINJ1 function. Endogenous NINJ1 forms heterogeneous ring-like structures during pyroptosis, consistent with in vitro observations. The study highlights a cookie-cutter mechanism for NINJ1-mediated membrane rupture, distinct from pore formation by GSDMD. NINJ1 oligomers cut membrane patches and release them, leading to membrane damage and cell death. The findings suggest that NINJ1 plays a critical role in pyroptosis and other lytic cell death pathways through a unique mechanism of membrane rupture.NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. The study reports the cryo-EM structure of a NINJ1 oligomer, revealing its role in membrane fragmentation. NINJ1 forms rings and filaments, and its oligomerization leads to membrane rupture by cutting and releasing membrane disks. The structure shows that NINJ1 has amphipathic and transmembrane helices, with kinked regions important for function. The oligomer has a concave hydrophobic side facing the membrane and a convex hydrophilic side formed by the amphipathic helices. NINJ1 can encircle and release membrane disks, consistent with membrane fragmentation. Live cell and super-resolution imaging show ring-like structures on the plasma membrane and released into the culture supernatant. Released NINJ1-encircled disks contain membrane, indicating NINJ1-mediated membrane rupture differs from gasdermin-mediated pore formation. NINJ1 forms rings and is released during pyroptosis, with live cell imaging revealing NINJ1 rings in the culture media. Mutational analyses confirm the importance of Gly residues at the TM kinks and positively charged residues for NINJ1 function. Endogenous NINJ1 forms heterogeneous ring-like structures during pyroptosis, consistent with in vitro observations. The study highlights a cookie-cutter mechanism for NINJ1-mediated membrane rupture, distinct from pore formation by GSDMD. NINJ1 oligomers cut membrane patches and release them, leading to membrane damage and cell death. The findings suggest that NINJ1 plays a critical role in pyroptosis and other lytic cell death pathways through a unique mechanism of membrane rupture.