Abstract: Objective: To investigate the anti-glioma effect and mechanism of AND on human glioma U251 cells via network pharmacology, bioinformatics and in vitro experiments. Methods: Network pharmacology was used to predict glioma-related targets based on the structure of AND. The hub genes were selected by bioinformatics, analyzed by gene ontology (GO) molecular function and kyoto encyclopedia of genes and genomes enrichment analysis (KEGG) to determine their related signaling pathways. Molecular docking was conducted to study the binding activity of AND with the above targets. Finally, gene set enrichment analysis (GSEA) was performed, analyzing the expression differences of hub genes and the Kaplan-Meier survival curve by comparing glioma samples with normal samples. On the above basis, the effects of AND on the growth and morphological changes of human glioma U251 cells were observed, the effects of AND on the PI3K/AKT pathway were detected. Results: A total of 40 glioma related targets of AND were identified by network pharmacology, and 10 hub genes were screened out by bioinformatics method. AND had high binding activity to hub genes. GO and KEGG enrichment analysis results showed that the hub genes were related to biological processes such as the regulation of neuronal apoptosis, reactive oxygen metabolism, and were related to PI3K/AKT signaling pathways. GSEA enrichment analysis suggested that PI3K/AKT-mTOR and other signaling pathways were significantly up-regulated in glioma cells. Among the hub genes, HSP90AA1, HSP90AB1, RHOA, CDK2 and MET showed significant differences between glioma samples and normal samples. Kaplan-Meier survival curve showed that low expression of genes like BCL2L1, CDK2, SOD2 and MET had higher survival rates. In the in vitro studies, AND inhibited the growth of U251 cells in both time-dependent and dose-dependent manners. After the administration of AND, U251 cells became round and shriveled, which significantly decreased the expression of p-PI3K, p-AKT, p-PI3K/PI3K, p-AKT/AKT to the PI3K/AKT pathway. Conclusion: AND can inhibit the proliferation of human glioma U251 cells, which mechanism is related to the PI3K/AKT signaling pathway.