Abstract: Background Previous studies have shown that Duloxetine (DUL) exhibits cytotoxicity in lung and pancreatic cancer cells, but its effects and mechanisms in GBM remain unclear.Objective To investigate the anti-tumor effects of Duloxetine on GBM and its potential mechanisms. Methods In this study, GBM cell lines U87 and U251 were treated with different concentrations of duloxetine (10, 20, and 30 μmol/L), with untreated control groups and a 300 μmol/L temozolomide (TMZ) treatment group as controls. CCK-8 and Transwell assays were used to evaluate the effects of Duloxetine on cell proliferation, migration, and invasion. Flow cytometry was used to analyze changes in the cell cycle. Potential mechanisms were explored by transcriptome sequencing. The expression of S-phase kinase-associated protein 2 (Skp2), p21, and p27 at both mRNA and protein levels was measured using qPCR and Western blotting. Molecular docking was performed using Autodock Vina to simulate the binding affinity between Duloxetine and the Skp2 protein. Results Duloxetine significantly inhibited GBM cell proliferation, migration, and invasion (P < 0.05). In U87 cells, the inhibition rates at 10, 20, and 30 μmol/L of Duloxetine were 18.06%±3.91%, 47.37%±1.42%, and 64.85%±0.90%, respectively (P < 0.001). Compared with the control group, Duloxetine treatment markedly increased the proportion of U87 cells in the G0/G1 phase (64.76%±1.2%, 72.18%±1.22%, and 79.03%±2.25% vs 55.72% ± 0.91%, P < 0.001). RNA-seq and validation experiments demonstrated that Duloxetine downregulated Skp2 mRNA and protein expression while upregulating p21 and p27 mRNA and protein levels (P < 0.05). Molecular docking revealed that Duloxetine exhibited high binding affinity with Skp2 (-7.4 kcal/mol).Conclusion Duloxetine inhibits GBM cell proliferation, migration, and invasion and induces G0/G1 cell cycle arrest by regulating the Skp2-p21/p27 signaling pathway, providing a new perspective for GBM treatment.