The most malignant type of tumor in the brain is high-grade gliomas. Glioblastoma (GB), a grade 4 glioma, has the lowest 5-year survival rate and is associated with poor prognosis. An important signaling pathway involved in the pathogenesis of GB is the mammalian target of rapamycin (mTOR). Therefore, our study aimed to investigate how exosomes obtained from GB cells applied with different doses of hesperidin (HSP) affect miR- 9 and change the PDK1/AKT/mTOR pathway. For this purpose, T98G cells were treated with different doses (5, 10, 25, and 50 µg/mL) of HSP in combination with temozolomide (TMZ- 10 µg/mL). At the end of 24 h, cell viability, flow cytometry, and biochemical tests were performed. Additionally, exosomes were isolated from cells belonging to the control, TMZ, and high-concentration TMZ-HSP groups. miR- 9, PDK1, PTEN, AKT- 1, Bax, Bcl- 2, and Caspase 3 genes were expressed in both application groups and exosomes belonging to these groups. HSP was found to reduce the viability of GB cells significantly. The viability was significantly reduced, especially in the TMZ-HSP 50 µg/mL group. Depending on the dose, there was a significant increase in the LDH level and oxidative stress level. The apoptosis level was approximately 26% in the TMZ-HSP 50 µg/mL group. Along with all this, gene expressions changed at the exosomal level, and miR- 9 and miR- 146 levels increased. Similarly, it changed the expression of proteins related to the PDK1/AKT/mTOR signaling pathway at the exosomal level (p < 0.05). In conclusion, the TMZ-HSP combination showed anticancer effects in T98G cells, influenced exosome profiles, and appeared non-toxic and potentially beneficial to healthy cells, highlighting its potential therapeutic value.