The purpose of the present study was to overcome resistance to imatinib (IM) by combining it with roscovitine (ROSC) and to investigate whether or not midkine (MK) had an effect on this combination in the treatment of glioblastoma (GBL). Human T98 GBL cells were used to evaluate the effects of IM (10 mu M), ROSC (200 mu M) and their combination on the cell proliferation index, apoptotic index, the apoptotic protein and anti-apoptotic protein levels, and ultrastructure. All applications decreased the cell proliferation index and increased the apoptotic index, but ROSC was the most efficient drug and the second most efficient drug was IM. Notably, ROSC increased anti-apoptotic proteins levels (PDGFR-alpha, AQP-4, hTERT), COX-1 activity and ribosome numbers. The effects of ROSC on hTERT, MK, AQP-4 and MRP-1 levels and COX-1 activity were reported for the first time. ROSC induced the highest increase in caspase-3 levels. Autophagy was not involved in the activity of ROSC in GBL spheroids. The combination of IM with ROSC showed an antagonist effect in the treatment of human GBL cells. The combination group decreased certain anti-apoptotic protein levels (PDGFR-alpha, EGFR, p-gp, MRP-1 and MK), cA MP levels, COX-1 activity and apoptotic protein levels (caspase-3). However, it induced the highest increase in hTERT levels and COX-2 activity. Ribosome numbers were much lower than those in the ROSC group and no autophagic vacuole was observed. In conclusion, more investigations are required to identify the key regulatory components that are responsible for this antagonism; however, the determination of this combination therapy as a failure therapy may be precautionary for oncologists in the treatment of GBL patients and potentially may contribute to the efficacy of new therapeutic regimens.