Objective: Risk of high grade gliomas is lower in young females and its incidence enhances after menopause suggesting likely protective roles of female hormones. Hormone replacement therapy (HRT) was widely employed to treat osteoporosis and some epidemiological studies showed that HRT regimes including progesterone analogs such as medroxyprogesterone acetate (MPA) decreased risk of glial tumors. Tibolone is a unique progesterone analog employed in HRT with tissue specific estrogenic effects and stimulates gene expressions very similar to those induced by MPA. Tibolone's pro-estrogenic effects occur particularly in bone and brain and both MPA and tibolone inhibit AKR1C enzymes, which involve in temozolomide chemoresistance. Hence, we aimed to investigate interactions between MPA, tibolone and temozolomide in modification of glioma cell growth and fine structure. Patients and Methods: For our studies, we have particularly chosen C6 rat glioma cell line due to several reasons: i) We previously showed that MPA reduced growth and induced procarbazine-sensitization in C6 cells; ii) temozolomide has a triazene-type molecular structure like procarbazine; iii) other groups previously showed that C6 glioma cell line is more resistant to temozolomide than human glioma cells; hence it may provide a native model of chemoresistance. Monolayer plating efficacy, soft agar colony growth, 3D-spheroid S-phase (as determined by BrdU-labeling) and electron microscopical analyses were performed to assess mutual interactions between MPA, tibolone and temozolomide. Results: MPA inhibited clonogenic growth of C6 glioma and this effect is augmented by both tibolone and temozolomide. MPA and tibolone inhibited DNA synthesis in C6 glioma spheroids to similar levels which can be achieved with temozolomide. Electron microscopical analyses revealed synergisms between MPA, tibolone and temozolomide involved mitochondria! proliferation, condensation, mitophagy and autophagy. Conclusions: MPA and tibolone shall be studied in further experimental models of glioblastoma in vitro and in vivo.