Research question: Is humanin present in the human ovary and follicular fluid? What relationship exists between humanin concentration in the follicular fluid and ovarian reserve and clinical outcomes after IVF and intracytoplasmic sperm injection (ICSI)? Design: Follicular fluid samples were collected from 179 patients undergoing their first IVF or ICSI cycle during oocyte retrieval. Ovarian tissues were collected from two patients undergoing surgery for ovarian cysts. Ovarian humanin localization was analysed using immunofluorescence staining. Expression of humanin in granulosa cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis. Follicular fluid humanin levels were evaluated with enzyme-linked immunosorbent assay. Relationships between follicular fluid humanin levels and ovarian reserve markers and clinical outcomes were analysed. Results: Strong humanin expression was found in the granulosa cells, oocytes and stromal cells of the ovary. Agarose gel electrophoresis of RT-PCR products showed rich humanin mRNA expression in human granulosa cells (119 bp). Follicular fluid humanin concentrations ranged from 86.40 to 417.60 pg/ml. They significantly correlated with FSH (r = -0.21; P < 0.01), LH (r = -0.18; P = 0.02), antral follicle count (r = 0.27; P < 0.01), anti-Mullerian hormone (r = 0.24; P = 0.03) and inhibin B (r = 0.46; P < 0.01) levels. Patients were subdivided into four groups according to follicular fluid humanin concentration quartiles (Q1-Q4). Patients in Q4 were more likely to achieve a pregnancy than Q1 (OR = 3.60; 95% 011.09 to 11.84). Conclusions: Humanin concentration in the follicular fluid was positively associated with ovarian reserve and clinical pregnancy rate.