Objective Otosclerosis is a widespread disease but the etiopathogenesis is still not fully understood. Hormonal factors especially estrogens are accused in recent years. The study aimed to evaluate the levels of G-protein associated membrane estrogen receptor-1 (GPER-1) and sex-hormones in patients with otosclerosis. Subject and methods The study included 60 people (30 otosclerosis patients, 30 control group). Serum sex-hormone (estradiol, progesterone, prolactin and total testosterone) and GPER-1 levels were measured in otosclerosis patients and compared with the normal population. For the otosclerosis group, air conduction and bone conduction thresholds and air-bone gaps were viewed from audiograms and the relationships between hearing and GPER-1 or sex-hormone levels were also investigated. Results Sex-hormone levels were not different between the groups. GPER-1 level was significantly lower in the otosclerosis group [3.1353 (0.76–8.21) ng/mL] than the control group [5.4773 (0.96–20.31) ng/mL] (p =0.017). Differential diagnosis with ROC analysis for the GPER-1 level was also significant (p=0.017). GPER-1 level was significantly lower for the females than the males in the otosclerosis group (p=0.043). Serum estradiol, progesterone, and prolactin levels were significantly higher (p=0.02, p =0.029 and p=0.019 respectively) and the GPER-1 level was significantly lower (p= 0.04) in the female patients compared to the female controls. There was no statistically significant relationship between GPER-1 or sex-hormone levels and hearing parameters. Conclusion GPER-1 level was lower in the otosclerosis patients compared to healthy volunteers and also lower in females than males in the patient group. Female sex-hormone levels were higher and GPER-1 level was lower in the female patient group than the female control group. Neither GPER-1 nor sex-hormone levels were not predictive of hearing levels. These findings indicate that sex-hormones especially estrogen and GPER-1 might have a potential role in the etiopathogenesis of otosclerosis. This is the first study in the literature that investigates the GPER-1 values in otosclerosis.