Objective To screen up-regulated genes and signaling pathways in glioma cells after knockdown of mammalian target of rapamycin (mTOR) gene, and to explore the efficacy of simultaneously blocking mTOR signaling pathway and its bypass activation pathways in inhibiting the growth of glioma cells. Methods Five human glioma cell lines U87, U251, U373, T98, and LN229 were selected to verify the protein expression of mTOR by Western blotting. U87 cells stably transfected with short hairpin RNA targeting mTOR gene were constructed, and the most significantly up-regulated genes and signaling pathways were screened in glioma cells after knockdown of mTOR gene by high-throughput sequencing. The pathway inhibitor with the highest inhibition rate was screened by cell drug sensitivity test. The cell activity was analyzed by cell counting kit 8. Results High mTOR expression cell line U87 was screened out, and the mTOR gene knockdown glioma cell model was successfully constructed. A total of 24 528 new transcripts and 1 906 differentially expressed genes were screened out by high-throughput sequencing. The top 12 up-regulated genes with high log₂|fold change|value were located in 9 bypass activation pathways. The inhibitor of signal transducers and activators of transcription 3 (STAT3) pathway with the highest inhibitory activity was screened out by drug sensitivity test. In vitro experiments showed that the STAT3 pathway inhibitor could increase the inhibition effect of mTOR gene knockdown on the proliferation of U87 cells (P<0.05). Conclusion Knockdown of mTOR gene in human glioma cells can activate the bypass signaling pathways. The inhibition effect can be effectively enhanced by combined use of inhibitors of bypass activation pathways.