In the study, to explore the anti-tumor effects and mechanisms of chlorpromazine (CPZ) and perphenazine (PPZ) combined with temozolomide (TMZ) on human glioma cell lines, we performed MTT assays to determine the growth inhibitory rate of CPZ, PPZ and TMZ in mono and combined treatments. The anti-tumor effects of CPZ and PPZ alone or in combination with TMZ were determined by colony formation, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) detection (JC-1). The expression level of p53 was detected by immunofluorescence assay. Furthermore, autophagy under different administrations was detected by flow cytometry and confocal imaging to explore the anti-tumor mechanism of CPZ and TMZ. Protein phosphatase 2A (PP2A), cancerous inhibitor of protein phosphatase 2A (CIP2A) and proto-oncogene protein (c-Myc) were detected by immunofluorescence assay, tumor stem cell markers (CD44, CD133) and aldehyde dehydrogenase (ALDH) were detected by flow cytometry to explore the anti-tumor mechanism of PPZ and TMZ. The results showed that after 72 h treatments of combinations, the values of half maximal inhibitory concentration (IC₅₀) of TMZ on U87 and U251 cells were reduced, and the ability of TMZ to induce apoptosis and cycle arrest was improved. In addition, the combination of CPZ and TMZ could induce an increased effect of autophagy via activating the relevant pathway of p53 gene in glioma cells. The combination of PPZ and TMZ increased the sensitivity of glioma cells to TMZ, and the underlying mechanism might be related to the inhibition of CIP2A/PP2A/c-Myc signaling pathway. In conclusion, CPZ and PPZ combined with TMZ, showed the significant synergistic effects in cancer treatment, which are the novel and potential therapeutic regimens providing a new treatment strategy for human glioma.