Human ovarian follicles develop a fluid-filled antrum when they reach a diameter of around 250 µm. The antrum contains follicular fluid (FF) composed of secretions from cells in the follicle and from circulation. The composition of FF undergoes massive changes in relation to follicular development. Previously, only FF from large pre-ovulatory follicles aspirated just prior to ovulation, has been analyzed by proteomics; resulting in a high proportion of plasma proteins being detected due to diffusion of proteins through the increasingly leaky basal membrane in larger follicles. We present the first proteomics analysis of FF from normal unstimulated human small antral ovarian follicles (diameter: 3-8mm) identifying the largest number of proteins reported in human FF to date: 2,050 proteins of which 1,151 were identified for the first time in human FF by mass spectrometry. Furthermore, an identified growth factor, midkine, was shown to impact follicular regulatory processes including enhancement of nuclear maturation of immature human oocytes.