The present study investigates the prognostic value of the FDX1 gene and its association with immune infiltration in gliomas. Gene expression profiles and corresponding clinical parameters of glioma patients were obtained from the Cancer Genome Atlas and Chinese Glioma Genome Atlas databases. In vitro experiments were also performed to validate its impact on malignant phenotypes of glioma cells. Kaplan-Meier analysis demonstrated that high FDX1 expression was associated with poor prognosis in glioma. Function and pathway enrichment for FDX1 predominantly demonstrated immunomodulatory function. In addition, the high-FDX1 expression group had higher Estimation of Stromal and Immune cells in malignant tumor tissues using Expression data, stromal, and immune scores (p<0.001). On evaluation of immunotherapy response, TIDE and dysfunction scores were higher in the low-FDX1 group, while the exclusion score demonstrated an opposite trend. In vitro tests showed that FDX1 silencing-induced inhibition of cell invasion and migration inactivated the nucleotide oligomerization domain (NOD)-like receptor signaling pathway by regulating PD-L1 expression. Notably, NOD1 expression was reversed in FDX1-knockdown cells after treatment with NOD1 agonists. In conclusion, FDX1 may play an important role in the diagnosis and treatment of gliomas. Regulating its expression may therefore help improve immunotherapy for these tumors.