Objective: To explore whether Neurotrophic factor 4 (NT4) could improve human follicular in-vitro- development and achieve mature and fertile oocytes. Design: Prospective study. Setting: Research institute. Population: Discarded ovarian tissues were collected from 7 patients (6 to 21 years old) underwent unilateral oophorectomy for fertility preservation. Methods: Isolated secondary follicles were cultured in vitro with or without NT4 in a matrix-free system individually. Main outcome measures: Follicle growth and survival were assessed by microscopy. Anti-Müllerian hormone (AMH), estradiol and progesterone levels were quantified in the medium. Oocyte marker was evaluated under confocal fluorescence microscopy. Oocyte mature and fertile potential were assessed by in-vitro maturation and intracytoplasmic sperm injection (ICSI) with donated sperm, separately. Results: In control condition, isolated follicles survived for 4-6 weeks with increased diameters over time ( P < 0.05), reaching a terminal diameter of 967.00 ± 41.1 μm with the induction of steroidogenesis and expression of oocyte marker. And 4/16 of alive follicles were matured in vitro, producing morphologically normal MII oocytes (120 ± 2.1 μm). The diameter and steroid hormone production were significantly higher in the NT4 group than the control group ( P < 0.05). A slightly increased efficiency of MII oocyte production in the NT4 group was also observed. MII oocyte obtained from the control group showed abnormal fertilization after ICSI, while progressed to available blastocyst in the NT4 group. Conclusions: NT4 promotes the proliferation, steroid hormone production of human secondary follicles cultured in vitro (including post- and pre-pubertal patients), thereby yielding mature and fertile oocytes.