Glioma is recognized as one of the most lethal and aggressive brain tumors. Although the standard-of-care treatment for glioblastoma (GBM) involves maximal surgical resection and temozolomide (TMZ) chemotherapy, the discovery of novel anti-tumor agents from nature sources is an effective strategy for glioma treatment. In this study, we conducted a screening process to identify the bisindole alkaloid melodinine J (MDJ) from Melodinus tenuicaudatus. We assessed its potency in overcoming TMZ resistance in patient-derived recurrent glioma strains, TMZ-resistant cell lines, and nude mouse tumor models of glioma cells. Our results first indicated that MDJ effectively inhibited malignancy and stimulated apoptosis in glioma. Mechanistic studies revealed that MDJ triggered deadly mitochondrial dysfunction and apoptosis by disrupting cross-organellar communication between the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). We also showed that high levels of TMX1 may promote malignancy of glioma by ER-mitochondria communications, bioenergetics efficiency, and tumor growth. Overall, our study proved that MDJ interfered the function of TMX1-mediated MAM networks, thereby overcoming the proliferation and chemo-resistance of glioma cells.