〔Abstract〕 To investigate the role mechanism of doublecortin (DCX) in glioma and screen DCX overexpression-related differentially expressed genes, so as to provide new targets for glioma targeted therapy.Methods Using rat glioma C6 cells as the target cells, LV-DCX-EGFP, LV-Cas9-Puro, and LV-Ctrl-EGFP lentiviruses were constructed. DCX overexpressing and control C6 cell lines were established through Cas9 dual- vector system transfection and screening. Transcriptome sequencing was used to obtain the differentially expressed gene profiles. iDEP and DESeq2 were applied to screen differentially expressed genes with the threshold of |log2FC|>2 and P<0.05. STRING and Cytoscape were utilized to construct protein-protein interaction networks and screen key genes. DAVID was employed for Gene Ontology (GO) enrichment analysis. BUSCA and UniProt were used to predict the subcellular localization of key genes. Receiver operating characteristic (ROC) curves were plotted to verify the diagnostic value of key genes. Enrichr was adopted to predict the regulatory networks of transcription factors and miRNAs. Real-time quantitative PCR (RT-qPCR) was performed to validate the expression of key genes. Results A total of 45 upregulated and 47 downregulated differentially expressed genes were screened, and 6 upregulated key genes including POU2F3 and 5 downregulated key genes including UBB were identified. GO enrichment analysis showed that upregulated genes were enriched in biological processes such as the positive regulation of lipid biosynthesis, whereas downregulated genes were linked to molecular functions including the regulation of alkaline phosphatase activity. Key genes were mainly distributed in the nucleus and cytoplasm. Except for RPS17, all key genes had an AUC value >0.7, indicating good diagnostic value. RT-qPCR validation showed that the mRNA expression levels of some upregulated genes (POU2F3,LPAR6, SREBF1) significantly increased (P<0.001), while the mRNA expression levels of some downregulated genes (UBB, ACTB, UBE2I) also significantly increased (P<0.001), which might be related to transcriptional and post-transcriptional regulation. Conclusion Differentially expressed genes and their regulatory networks related to DCX overexpression in glioma are successfully screened, providing a theoretical basis for revealing the role mechanism of DCX in glioma development and laying a foundation for the development of potential therapeutic strategies.