Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis after conventional treatment. Therefore, potential treatment strategies and novel therapeutic targets have gained increased attention. Vanilla planifolia is a source of vanilla flavoring due to its high vanillin content, and several studies have shown its antitumor activity. Our data revealed that an ethanol extract of vanilla planifolia stem (VAS) at 200 ng/μl significantly reduced cell viability and colony formation in GBM cells. VAS also induced autophagy, as shown by MAP1LC3 cleavage. Further RNA-seq analysis and MA plot identified 1972 upregulated differentially expressed genes (DEGs) and 2276 downregulated DEGs in GBM cells treated with 200 ng/μl VAS compared to the control. Protein-protein interactions were analyzed among DEGs with fold changes less than -3 and greater than 5, revealing 16 and 9 hub DEGs, respectively, correlated with other DEGs. qPCR experiments confirmed significant downregulation of mRNA expression for DHRS9, HOPX, AQP5, PCP4, RGS8, GNAT2, RLBP1, FA2H, TNMD, SKAP1, MATN1, IGFBP1, ELFN2, and C2CD4C. Additionally, the expression of IL36RN, CCL20, CCL5, CXCL10, HMOX1, MX2, RSAD2, IFI44L, and EGR1 was significantly upregulated. Furthermore, PAK6, another significantly downregulated DEG, correlated with the overall survival of GBM patients using the TCGA databank. Validation experiments confirmed that VAS significantly reduced the mRNA and protein expression of PAK6, which led to the abolition of cell viability and colony formation. These findings demonstrate that VAS reduces cell viability and colony formation, induces autophagy, and identifies PAK6 and other DEGs as potential therapeutic targets for GBM treatment.