Without effective treatment, glioblastoma is one of the deadliest cancers worldwide. The aim of the present study was to explore whether combinational immunotherapy is effective for treating malignant glioblastoma in vitro. The therapeutic efficacy of third generation anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR)-T cells alone and in combination with PD1 blockade was investigated for the treatment of malignant glioblastoma cells in vitro. Anti-HER2 CAR-T cells were prepared by transducing activated primary human T cells with lentiviruses which expressed third generation anti-HER2 CAR. The CAR-positive cell ratio was detected using flow cytometry. The expression level of CAR was detected by western blot analysis. The binding of anti-HER2 CAR-T cells to HER2(+) U251 glioblastoma cells was examined under a fluorescence microscope. The cytokine secretion of CAR-T cells induced by target cells was analyzed via ELISA. The cytotoxicity of anti-HER2 CAR-T cells alone or in combination with anti-programmed death-1 (PD1) antibody against HER2(+)/PDL1(+) U251 cells was examined using an LDH assay. The CAR-positive cell ratio and expression level of CAR in prepared CAR-T cells were both high enough. Anti-HER2 CAR-T cells could specifically bind to U251 cells. The IL-2 and IFN-gamma secretion of CAR-T cells increased after being co-cultured with U251 cells, and further increased in the presence of anti-PD1 antibody. Anti-HER2 CAR-T cells displayed a potent cytotoxicity against U251 cells. In addition, the presence of anti-PD1 antibody further enhanced the efficacy of anti-HER2 CAR-T cells against U251 cells. The present results indicated that blocking PD1 immuno-suppression can increase the activation of CAR-T cells after they are activated by a targeting antigen. Third generation anti-HER2 CAR-T cells along with PD1 blockade have a great therapeutic potential for combatting malignant glioblastoma.