〔Abstract〕 To investigate the potential mechanisms of Xanthatin in inhibiting the proliferation of laryngeal squamous cell carcinoma (LSCC) cells by integrating network pharmacology and in vitro experiments. Methods The targets of Xanthatin were identified using databases such as PharmMapper, while disease-related targets for LSCC were obtained from databases such as DisGeNET. The overlapping targets between Xanthatin and LSCC were determined by intersecting these datasets. A protein-protein interaction (PPI) network was constructed based on the overlapping targets, and key targets were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the overlapping targets were performed using R software. A "Xanthatin-target-pathway" network was visualized using Cytoscape 3.8.0 software. The preliminary validation of the aforementioned results was performed using molecular docking and transcriptomics. The effects of Xanthatin on the proliferation of TU177 cells were assessed using CCK-8 and colony formation assays. Additionally, Western blot analysis was employed to measure the expression levels of PI3K, p-PI3K, Akt, and p-Akt proteins. Results A total of 159 overlapping targets between Xanthatin and LSCC were identified, and seven key targets, including AKT1, were screened. GO enrichment analysis yielded 2,455 entries, and KEGG enrichment analysis identified 172 pathways, such as the PI3K-Akt signaling pathway. Xanthatin exhibited favorable binding activity with the core target proteins of LSCC in molecular docking experiments. The transcriptomics results showed high consistency with the predictions from network pharmacology. CCK-8 and colony formation assays demonstrated that Xanthatin at concentrations of 1, 2, and 4 μmol/L significantly inhibited the proliferation of TU177 cells in a dose-dependent manner. The expression levels of p-PI3K and p-Akt proteins decreased with increasing concentrations of Xanthatin. Conclusion Xanthatin may exert an inhibitory effect on the proliferation of laryngeal squamous cell carcinoma (LSCC) cells by modulating the PI3K-Akt signaling pathway.