Background <p>Hyperoside (Hyp) exerts considerable inhibitory effects on non-small-cell lung cancer (NSCLC) cells. However, the underlying molecular mechanisms have not been fully elucidated. We aimed to explore the molecular mechanisms of Hyp in the treatment of NSCLC using a combination of network pharmacology and in vitro experiments.</p> Methods <p>Active targets of Hyp and NSCLC-related targets were identified using public databases. The therapeutic targets were predicted based on the intersection of drug and disease targets. A protein–protein interaction (PPI) network was constructed using STRING and Cytoscape, and key network modules and targets were identified through network topology analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using DAVID. Molecular docking between Hyp and therapeutic targets was performed using AutoDock Vina software. These results were further verified by in vitro experiments of A549 cell lines, including the CCK-8 assay, EdU staining, flow cytometry and western blotting.</p> Results <p>A total of 30 therapeutic targets of Hyp against NSCLC were identified. Analysis of the PPI network revealed a key network module and five key targets: MMP9, CASP3, MAPK1, ESR1, and EGFR. These targets were related to cell proliferation and migration, apoptosis, and oxidative stress and may play a role through the FoxO, MAPK, Rap1, Ras, and PI3K-Akt signaling pathways. Molecular docking results showed a strong binding affinity between Hyp and most therapeutic targets. Further experiments confirmed that Hyp inhibited proliferation and induced apoptosis of A549 cells by regulating the EGFR/ERK/FOXO1 signaling pathway.</p> Conclusions <p>Hyp may be a promising drug for treating NSCLC (especially lung adenocarcinoma), and its therapeutic mechanisms is closely related to the regulation of EGFR/ERK/FOXO1 pathway.</p>