Glioblastoma (GBM) is a highly aggressive primary brain tumor associated with poor prognosis and enhanced aerobic glycolysis (the Warburg effect), which facilitates rapid tumor growth and increases malignancy. Phosphofructokinase 2 (PFK2), also referred to as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB), is a critical regulator of glycolysis and has been linked to tumorigenesis; however, the specific contributions of its isoforms to GBM pathobiology remain insufficiently defined.This study aimed to clarify the isoform-specific function of PFKFB4 in GBM by evaluating its expression pattern, clinical significance, and impact on tumor glycolytic flux, proliferation, and malignancy, as well as to investigate the therapeutic potential of selectively targeting PFKFB4.We examined the expression profiles of PFK2 isoforms in GBM using publicly accessible datasets. The clinical relevance was determined by conducting survival analysis using The Cancer Genome Atlas (TCGA) database. Protein expression patterns were identified with the Human Protein Atlas. PFKFB4 expression in GBM cell lines was validated using quantitative real-time PCR and immunoblotting. Functional roles of PFKFB4 were further assessed by employing the selective inhibitor 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN). Effects of 5MPN on cell viability, proliferation, glucose uptake, lactate production, migration, invasion, and protein expression (c-Myc, cyclin D1, MMP-2, MMP-9) were evaluated using WST-8 assay, colony formation, 2-NBDG uptake, lactate assay, wound healing, transwell assays, and immunoblotting.We found that PFK2 isoforms-notably PFKFB4-are markedly overexpressed in GBM tissues and cell lines, with elevated PFKFB4 expression associated with worse patient prognosis. Pharmacological inhibition of PFKFB4 using the selective small-molecule inhibitor 5MPN reduced the Warburg effect, cell proliferation, and colony formation in GBM cells, while having minimal effects on normal human astrocytes. Additionally, PFKFB4 inhibition diminished GBM cell migration and invasion, coinciding with a reduction in the expression of matrix metalloproteinases MMP-2 and MMP-9.These results underscore the oncogenic role of PFKFB4 in enhancing the Warburg effect and promoting malignant phenotypes in GBM, and support its potential as a therapeutic target for GBM treatment.© 2025. The Author(s) under exclusive licence to The Genetics Society of Korea.