Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.