Aim:St. John\'s Wort Oil (JWO) has a sedative property and it is used traditionally for the treatment of depression, neuralgia and excitability. JWO has been shown to have anticancer activity via apoptosis in glioblastoma cells. However, information on whether JWO is effective on the autophagy mechanism in glioblastoma is still not known. So, the current study was the first to search the autophagy mechanism T98 glioma cells. Material and Methods:Three groups were created with T98 human glioblastoma cells; Group 1: T98 glioma cells with no treated (Control group). Group 2: T98 glioma cells treated with 3 µl/ml JWO. Group 3: T98 glioma cells treated with 6 µl/ml JWO. The cell proliferation, oxidative stress, types of cell death were studied at IC50 dose of JWO.Results:The proliferation of glioma cells was inhibited in 5.296 µl/ml dose. JWO induced apoptosis in T98 glioma cells in comparison with the control and there was statistically significant difference (p<0.001). Apoptosis was analyzed via TUNEL method and results were checked by flow cytometry. We also investigated the effects of JWO on autophagy in T98 glioma cells by immunostaining LC3-II and MDC fluorescent stainings. The differences between JWO treated and control group were notably significant (p<0.001). The immunofluorescence staining resultsof LC3-II was confirmed by Western blotting analysis. Conclusion:JWO seems to be an effective treatment agent for glioblastoma. Not only does it induce apoptosis via oxidative stress but also affects the autophagy. The use of JWO in combination with other treatment options may increase the efficacy of treatment.