The effective treatment strategies for glioblastoma (GBM) are still limited at present. Identifying therapeutic targets in GBM and developing corresponding drugs are unmet needs. Here, we find that VCP is highly expressed in GBM cells and correlates with glioma malignancy. V8 is derived from Wogonin analogues, which bind to VCP to inhibit GBM growth. V8 inactivates its ATPase activity, and induces protein aggregates in cytoplasm and mitochondria. Abnormally accumulated VCP on mitochondria induced by VCP inhibitor further recruits PRKN, leading to the co-localization of mitophagy receptors with mitochondria to initiate mitophagy. However, inhibiting VCP also disturbs lysosomal pH, preventing the degradation of abnormal mitochondria. As a consequence, mitochondria with protein aggregates accumulate and release excessive mt-ROS which lead to the demise of GBM cells. In conclusion, VCP not only maintains mitochondrial proteostasis, but also keeps the integrity of lysosome to clear damaged mitochondria by mitophagy. Targeting VCP with its inhibitors, such as V8, causes mitochondria dysfunction, effectively suppresses the viability of GBM and may represent a potential strategy for GBM treatment.