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Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia , L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism. • Mutated Loxosceles recombinant Phospholipases-D are non-toxic proteins with antigenic properties. • Vaccination protocols prevented mouse lethality induced by Loxosceles intermedia venom. • Vaccination protocols prevented signals of cutaneous loxoscelism in rabbits caused by Loxosceles venoms. • Serum of vaccinated animals neutralized cutaneous loxoscelism induced by Loxosceles intermedia venom. • Results evidence these non-toxic Phospholipases-D as potential antigens to develop protective vaccines for Loxoscelism. [ABSTRACT FROM AUTHOR]