Summary The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild‐type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3‐kinase (PI3‐K)/AKT pathway‐focused gene profiles were examined by real‐time polymerase chain reaction‐based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen‐activated protein kinase kinase inhibitor U0126. The dual PI3‐K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal‐regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.