Objectives: PTEN-Long is a translational variant of phosphatase and tensin homolog (PTEN). This study aimed to assess the effect of PTEN-Long on the biological characteristics of glioma cells and related mechanisms. Methods: A vector stably expressing PTEN-Long was established and transfected into cells, serving as the overexpression group, while a set of empty vectors served as the negative control group. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and western blot were used to detect the expression of PTEN-Long and phosphatidylinositol 3-kinase, Protein kinase B, andnuclear factor-kappa B (PI3K-AKT-NF-kappa B). Cell proliferation was assessed with the Cell Counting Kit 8 (CCK8) assay, migration through the scratch test, and invasion by the transwell chamber assay. Cell cycle analysis was performed using flow cytometry. The volume and weight of subcutaneous tumors in nude mice were also evaluated. Results: PTEN-Long expression led to downregulation of p-Akt, NF-kappa B p65, p-NF-kappa B p65, and Bcl-xl, and up-regulation of I kappa B alpha. In addition, it inhibited glioma cell proliferation, induced cell cycle arrest in the G0/G1 phase, and reduced cell migration and invasion. Moreover, PTEN-Long inhibited the growth of subcutaneous glioma in nude mice. Conclusions: PTEN-Long inhibits the proliferation, migration, and invasion and induces apoptosis in glioma cells by inhibiting PI3K-AKT-NF-kappa b signaling, implying that PTEN-Long may be a new target for glioma treatment.