MicroRNAs (miRs) are known to repress target genes at posttranscriptional level and play important roles in the maturation of cells. However, the expression profiles of miRs during follicular maturation have not been fully elucidated. This study was designed to investigate the expression profiles of miRs in murine follicles according to human chorionic gonadotropin (hCG) treatment and vitamin C status during in vitro culture. Ovaries were removed from the 12-day-old wild-type and vitamin C-deficient (L-gulonogammalactone oxidase knockout, Gulo-/-) C57BL6 mice. Preantral follicles were isolated and cultured in 20 µL droplets of culture media supplemented with follicle-stimulating hormone and luteinizing hormone (FSH + LH). After their full maturation, follicles were divided into 2 groups: with and without hCG treatment. Real-time polymerase chain reaction (PCR) was performed using oocytes and granulosa cells (G-cells) to evaluate the miRs known to be expressed mainly in the mouse ovary. After the addition of hCG, miR profiles showed divergent changes between oocytes and G-cells. These profiles significantly differed from those of hCG(-) group. Compared to wild type, Gulo-/- mice showed altered miR profiles in matured oocytes and G-cells. Conclusively, hCG supplementation and vitamin C status alter the miR expression profiles in oocytes and G-cells during in vitro growth of murine follicles.