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Gliomas are primary brain tumors that develop from glial cells within the central nervous system and are among the deadliest human cancers. Glioblastoma (GBM) is the most malignant form of glioma. NLRX1 is an innate immune pattern recognition receptor that exhibits tumor-suppressive or tumor-promoting effects that may be cancer type- and context-dependent, aided by differences in the microenvironment. Here, we report that NLRX1 is differentially expressed in microglia, astrocytes, GBM cell lines, and glioma patient tissues. siRNA-mediated silencing of NLRX1 induces metabolic stress in GBM cells, as observed by an increased number of tunneling nanotubes (TNTs) formation between GBM cells and decreased expression of autophagy markers. Moreover, silencing of NLRX1 decreases the ability of the GBM cell lines, LN-229 and LN-18, to proliferate and migrate. si-NLRX1 GBM cells exhibit attenuated ability to generate 3D spheroids. In summary, our findings indicate that NLRX1 positively regulates GBM pathophysiology by supporting GBM cell metabolism, proliferation, migration, and anchorage-independent growth. We believe our understanding of NLRX1 in GBM pathophysiology paves the way for potential development of GBM-targeting therapeutics that may delay disease progression and/or improve survival.