Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.