Dermonecrosis resulting from Loxosceles spider envenomation, primarily driven by the enzyme sphingomyelinase D (SMase D), is characterized by severe inflammation and nonhealing wounds. SMases can be classified as Class I or II based on their structural characteristics. Class I exhibits greater dermonecrotic activity than Class II; however, the intracellular mechanisms responsible for this difference remain poorly understood. The differential transcriptomics analysis of human keratinocytes treated with each toxin revealed that Class I primarily activates pathways associated with proteolytic activity and apoptosis. In contrast, Class II uniquely upregulates key genes, including PIM-1, MCL-1, PAI-1, p21, and c-FOS, which support cell survival and inhibit apoptosis. These pathways also facilitate tissue repair and keratinocyte proliferation during wound healing, particularly through signaling mechanisms involving Substance P and VEGF-A. RT-qPCR confirmed these findings, with protein level evaluations indicating the sustained upregulation of VEGF-A exclusively in keratinocytes treated with Class II. We identified Substance P and VEGF-A as potential therapeutic targets for managing cutaneous loxoscelism, providing valuable insights into the cellular mechanisms underlying the distinct toxic effects of the two SMase D isoforms. By elucidating these pathways, this study enhances our understanding of loxoscelism’s pathophysiology and highlights strategies for therapeutic intervention in dermonecrotic injuries caused by spider venom.