OBJECTIVE: This study predicted and verified the effects of Jujuboside A (JuA) on the proliferation and migration ability of glioma cells to developing new therapies for glioma treatment. METHODS: The druggability of JuA was determined by using cheminformatics. Network pharmacology was used to analyse common targets, biological function and metabolic pathways of JuA against glioma. The core targets of JuA against glioma were validated by using molecular docking. The biological functions of JuA were verified by in vitro experiments. RESULTS: Cheminformatics results showed that JuA is possible to be a drug. Network pharmacology revealed 294 shared targets between JuA and glioma, which were associated with proliferation, migration, and multiple signalling pathways. A total of 16 core targets related to the signalling pathways were verified by molecular docking. The in vitro experiments showed that JuA could inhibit cell proliferation and migration, decrease cell numbers and alter cell morphology. CONCLUSION: The results of network pharmacology and in vitro experiments indicate that JuA has significant toxic effects on glioma cells, and can play a therapeutic role in treating glioma by inhibiting the proliferation and migration of glioma cells.