Background: Gender differences in brain physiology and gender differences in pathology are usually recognized, but often are disregarded in clinical and experimental studies, resulting in numerous inaccuracies in data interpretation. The purpose of this study was to analyze levels of neurosteroid hormones in mitochondria of the cerebral cortex cells in C57BL/6 mice with subcutaneous B16/F10 melanoma growing in presence of chronic neurogenic pain (CNP). Methods: The study included male and female C57BL/6 mice (n = 336) aged 8 weeks initially weighing 21-22 g. Experimental groups were: intact animals; controls with a CNP model created by bilateral sciatic nerve ligation; comparison group 3 weeks after subcutaneous inoculation of 0.5 ml suspension of B16/F10 melanoma cells diluted 1:10; main group 3 weeks after subcutaneous melanoma growth in presence of CNP (CNP+B16/F10). Levels of estradiol (pg/g protein), estrone (pg/g protein) (DBC, Canada); progesterone (ng/g protein), total and free testosterone (pg/g protein) (XEMA, Russia) were measured in mitochondrial samples by ELISA (Tecan Infinite F50 analyzer, Austria). Results: Levels of estradiol in intact females were 3.1 times higher than in males, while estrone, progesterone, total and free testosterone were lower by 6.4, 2.7, 2.0 and 2.5 times, respectively. Only in females with CNP estradiol decreased by 3.4 times, compared to intact values, and estrone increased by 1.7 times (p<0.05), testosterone by 3.9 times. Estradiol in animals with B16/F10 decreased compared with intact values by 3.1 times in females and by 1.5 times in males (p<0.05). Females with B16/F10 showed the highest levels of progesterone exceeding intact values by 3.1 times. In the group with CNP+B16/F10, females showed lower levels of estradiol and estrone (by 1.4 times, p<0.05) and free testosterone (by 1.6 times, p<0.05), compared to the levels in CNP; on the opposite, males had 1.5 times (p<0.05) higher estrone and 2.9 times lower progesterone. Conclusions: Low levels of estradiol involved in the protective mechanism of neurosteroids were the dominant factor in mitochondria of cerebral cortex cells in females with CNP and malignant neoplasms. Males did not demonstrate such dominant factor. In animals with CNP+B16/F10, the response nature of cerebral cortex mitochondria changed: all defense mechanisms in the brain of females were suppressed by the 3rd week of tumor development in presence of pain. In males, mitochondria of cerebral cortex cells were more resistant to the influence of two pathologies, and only few changes in the neurosteroid status were recorded.