The lethal factor in stonefish venom is Stonustoxin (SNTX), a cytolytic protein that induces profound hypotension and cardiovascular collapse in mammals. Here, we make the unexpected finding that SNTX is a pore forming member of a hitherto undiscovered third branch of the Cholesterol Dependent Cytolysin / Membrane Attack Complex/Perforin (CDC/MACPF)-superfamily. Our data suggest that initial SNTX interaction with target membranes is mediated by two C-terminal PRYSPRY domains that are most closely related to the lipid and protein binding PRYSPRY domains of Tripartite motif (TRIM) immune proteins. Two N-terminal CDC/MACPF domains are arranged in a pre-pore like assembly; these data provide unique, and broad, high-resolution insight into how SNTX and other CDC/MACPF proteins assemble into pores. Finally, our analysis reveals that SNTX-like CDCs are distributed throughout eukaryotic life, and likely play a broader, and possibly immune-related function outside venom.