The aim of this study was to evaluate the effects of ovarian tissue vitrification and two-step in vitro culture on the metaphase II (MII) oocyte reactive oxygen species (ROS) level, mitochondrial transcription factor A (TFAM) expression and succinate dehydrogenase (SDH) activity. After collection of neonatal mouse ovaries, 45 ovaries were vitrified and the others (n = 45) were considered as control. All ovaries were cultured for seven days, and their isolated preantral follicles were cultured in three-dimensional culture system. After 12 days in vitro culture, the follicular development and oocyte maturation were evaluated and compared in vitrified and non-vitrified ovaries. The collected MII oocytes were inseminated with capacitated spermatozoa. The fertilization, embryonic development, ROS level, TFAM gene expression and SDH activity of oocytes were assessed and compared. There was no significant difference between morphology and percentage of normal follicles between vitrified and non-vitrified ovaries at the beginning of culture. The follicular development and hormone level in the vitrified group was significantly lower than non-vitrified group and the ROS concentration in the vitrified group was significantly higher than non-vitrified group after one-week organ culture. After follicular culture, there was no significant difference in follicular development, oocyte maturation, fertilization rate, TFAM gene expression, ROS level and mitochondrial SDH activity between vitrified and non-vitrified groups. This study showed that mouse ovarian tissue vitrification influenced the follicular development through increase in ROS level during organ culture but these harmful effects of vitrification method may be recovered during the follicular culture period. Thus, vitrification and ovarian organ culture method should be improved.