This study aims to explore the mechanism of Ligusticum chuanxiong in the treatment of glioblastoma (GBM) by network pharmacology and in vitro cell experiments. The TCMSP, DrugBank, DisGeNET, GeneCards and OMIM databases were used to predict the active components and target genes of L. chuanxiong, as well as to obtain disease-related targets of GBM. The intersection of these targets was identified as the potential targets of L. chuanxiong against GBM. GO functional annotation and KEGG pathway enrichment analysis were performed using DAVID v2021q4. The STRING 11.0 platform and Cytoscape 3.8.2 software were used to construct a protein-protein interaction network of potential targets, screen core targets and validate their binding affinity through molecular docking. The effects of L. chuanxiong extract on the proliferation and invasion of U251 cells were detected using the CCK-8 assay and Transwell. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were used to verify the effects of L. chuanxiong extract on the expression of related target genes and proteins. Network pharmacology analysis identified 69 potential targets of L. chuanxiong against GBM and five core targets were screened out. These targets were mainly involved in pathways related to cell proliferation and apoptosis, cancer-related pathways and metabolic pathways. Molecular docking showed the core targets had good binding affinity with active components of L. chuanxiong, such as senkyunolide, ethyllinoleate and sitosterol. In vitro cell experiments showed that, compared with the control group, L. chuanxiong extract significantly reduced the viability of U251 cells (P<0.05), inhibited their invasion, increased the mRNA and protein expression of cysteinyl  aspartate specific proteinase 3 (Caspase-3),  decreased the mRNA and protein expression of signal transducer and activator of transcription 3 (STAT3) and epidermal growth factor receptor (EGFR) (P<0.05). In summary, L. chuanxiong exerts anti-GBM effects by influencing core targets such as Caspase-3, STAT3 and EGFR, thereby regulating pathways related to cell proliferation and apoptosis, cancer-related pathways and metabolic pathways.