Gasdermins (GSDMs) are pore-forming proteins that mediate pyroptosis and contribute to inflammatory and cancer-related processes. Although GSDME shares structural similarity with other gasdermins, its activation and pore assembly mechanisms remain incompletely defined. Here we determine high-resolution cryo-electron microscopy structures of 27- and 28-fold human GSDME pores at 3.64 Å and 3.58 Å resolution. The structures reveal conserved structural architecture together with distinct features, including an extended transmembrane β-barrel and a comparatively compact membrane-engagement geometry. Structure-guided mutagenesis identifies lipid-binding and oligomerization interfaces required for pore formation. We further demonstrate that caspase-3 activates GSDME through direct recognition of a DMPD tetrapeptide motif within the interdomain linker, independently of the GSDME C-terminal domain. Following proteolytic activation, S-palmitoylation of GSDME N-terminal domain enhances pore-forming efficiency, with Cys180 serving as the primary functional site. Together, these findings establish a coordinated structural and regulatory framework in which proteolytic licensing and lipid modification sequentially control GSDME pore formation.