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Glioblastoma is a highly invasive primary brain tumor with a poor prognosis, highlighting the need for new therapeutic strategies. Toxins derived from Macrodactyla doreensis have attracted attention for their potential anticancer activity. This study evaluated the anticancer and cytotoxic effects of M. doreensis crude venom on two commonly used glioblastoma cell lines (U251 and LN229), which mirror the phenotype of primary tumors. Cell viability and proliferation were assessed using the CCK-8 assay and colony formation assay, while cell migration and invasion capabilities were detected via wound healing assay and Transwell assay. Annexin V/PI staining and PI-based cell cycle analysis indicated that the crude venom significantly induced cell apoptosis and caused S-phase arrest. Proteomic analysis combined with GO and KEGG enrichment analyses as well as bioinformatics approaches showed that M. doreensis crude venom inhibits glioblastoma cell proliferation by downregulating the expression of CDK2, RRM2, and CHEK1, thereby hindering cell cycle progression and regulating the p53 signaling pathway. Notably, the downregulation of these key glioblastoma-related target genes was validated by qPCR. In addition, network pharmacology analysis indicated that several peptide families present in the sea anemone crude venom, including ShK peptides, inhibitor cystine knot (ICK) peptides, and EGF-like peptides, exhibit notable antitumor potential. Combined with AlphaFold2-based structural modeling and molecular docking, these analyses further elucidated the potential molecular mechanisms underlying their interactions with key targets, such as MD-381 with RRM2, MD-322 with CDK2, and MD-429 with CHEK1. Collectively, these findings highlight the therapeutic potential of M. doreensis crude venom and lay a foundation for the subsequent isolation of novel peptides and their further development in glioblastoma treatment.