AbstractBackgroundVesicle trafficking is a highly important process in numerous human diseases, especially in the central nervous system dysfunctions. However, as a key component of vesicle trafficking‐related genes (VRGs), Cornichon family AMPA receptor auxiliary protein 4 (CNIH4) has not been systematically elucidated in glioma so far.MethodsDifferentially expressed VRGs were selected using molecular signatures database (MSigDB), The Cancer Genome Atlas (TCGA), and Genotype‐Tissue Expression (GTEx) mRNA expression profiles. Further exploration of CNIH4 was determined using LASSO‐Cox regression algorithms. Then Kaplan–Meier (K‐M) plotter, receiver operating characteristic (ROC) curves, and multivariate Cox regression analyses were utilized to assess the independent significance of CNIH4 in the CGGA validation cohort. Functional exploration was performed with Gene Set Enrichment Analysis (GSEA) and then verified using a series of functional experiments in glioma cells. Finally, the consensus clustering algorithm was applied to identify clusters in glioma samples. After that, differences in prognosis, the tumor immune microenvironment (TIME), and therapy response were evaluated between clusters.ResultsCNIH4 was shown to be overexpressed in malignant glioma variants and was frequently observed in GCSs and TMZ‐resistant cell lines. Higher CNIH4 levels were significantly related to poor outcomes and positively correlated with adverse clinicopathological characteristics. Survival analyses revealed CNIH4 as an independent risk factor that outperformed traditional measures. Enrichment analysis indicated that overactive CNIH4 significantly gathered in stem cell processes. Furthermore, functional assays of silencing CNIH4 expression suppressed stem cell‐like properties in vitro and inhibited tumorigenicity in vivo. Finally, the CNIH4‐enriched subgroup negatively modulated immunotherapeutic response and reflected lower chemotherapy sensitivity for glioma patients.ConclusionOur study identified CNIH4 as a potential VRG that regulates tumor stemness, microenvironment immunity, and chemotherapy sensitivity. It may serve as a novel prognostic factor and a promising target against glioma therapy.