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As a black widow spider, <i>Latrodectus tredecimguttatus</i> has poisonous components not only in venomous glands but also in eggs. Our previous work had carried out a transcriptome analysis of the spider eggs in an attempt to probe into the molecular basis of the egg toxicity. A proteinaceous toxin, named Latroeggtoxin-V, was mined from the identified transcriptome. In this study, the gene of Latroeggtoxin-V was cloned and heterologously expressed, and the anticancer activity of the recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was characterized. Activity assay found that rLatroeggtoxin-V could selectively act on breast cancer line MDA-MB-231 cells, not only arresting their cell cycle, inhibiting their proliferation and migration, but also inducing their apoptosis. Bioinformatics analysis suggested that Latroeggtoxin-V belongs to the ATPase inhibitor protein family and the further activity assay showed that the rLatroeggtoxin-V inhibited the activity of the Na⁺/K⁺-ATPase in MDA-MB-231 cells in a concentration-dependent manner, suggesting that the anticancer activity of Latroeggtoxin-V is based on its affecting the ion transport and receptor functions of Na⁺/K⁺-ATPase. The present work not only laid the foundation for the utilization of Latroeggtoxin-V in the anticancer drug development and the related fields, but also provided a new paradigm for exploration of the proteinaceous toxins under the direction of transcriptomics and bioinformatics.