Envenomation by recluse spiders (genus Loxosceles) can cause dermonecrosis, hemolysis, and even death. Despite presenting a significant medical challenge in the Americas, especially in Brazil, the molecular mechanisms of toxicity are not well understood. The toxic component of Loxosceles venoms is a family of phospholipase D (PLD) enzymes that cyclise sphingolipid and lysophospholipid substrates in cell membranes. Structures of phospholipase D have been previously solved in the unbound form, however, experimental data for substrate binding and catalysis is lacking. We have solved the crystal structure of a phospholipase D toxin at 1.85 Å bound to substrate and product lipids in the active and noncatalytic sites. The structure confirms our previously proposed catalytic mechanism, offers possible mechanisms of allosteric activation, and, surprisingly, reveals interactions between protein subunits and lipid molecules that may inform on the membrane interface. The structure advances our understanding of the molecular basis of loxoscelism and provides a framework for the development of therapeutics to prevent or delay the effects of Loxosceles envenomation.