Abstract Glioblastoma is a lethal brain tumor. Despite aggressive treatment, the median survival time is 6 to 12 months. These tumors acquire resistance against many therapeutic regimens. There is a need to screen for potential new drugs and drug combinations. Cucurbitacin B (CuB), a compound obtained from plants, and evodiamine (Evo), a supplement, have shown promising results in various neoplasms but have not been well studied in glioblastoma and especially, as a combination treatment. This study investigated the combined effects of CuB and Evo on U-87MG human glioma cell line. Results demonstrated that CuB and Evo individually decreased the U-87MG cell viability in time and dose dependent manners. The effect was significantly pronounced with the combined treatment. The combination treatment of CuB and Evo induced cellular apoptosis most likely by interfering with the PI3K/AKT and JNK/ERK pathways. In addition, the flow cytometry analysis showed that the combination treatment arrested the cell cycle at the G2M phase and inhibited cell propagation. Moreover, CuB and Evo combined effect successfully inhibited cellular migration. The cytotoxic effect of combination treatment of CuB and Evo was three times lower in neuronal PC-12 cells compared to U-87MG cells. Overall, a combination of cucurbitacin B and evodiamine proved to be a superior potential anti-neoplastic treatment in U-87MG cells as compared to individual treatment, and may be considered as a potential adjuvant therapy for GBM. Citation Format: Vrutti V. Mehta, Kenza E. Benzeroual. The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2686.