Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.