Follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI-8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI-8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI-8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI-8 with reported FSH-FSHR hormone binding (FSHR(HB)) domain complex using ZDOCK algorithm revealed that the FRBI-8 binds to FSHbetaL2-FSHR(HB) binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI-8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI-8(D6A)) had a higher binding affinity than the native FRBI-8. In vitro radioreceptor assay with FRBI-8(D6A) showed 50% lower IC(50) compared with the FRBI-8, confirming the in silico observations. Thus, the study reveals that both FRBI-8 and FRBI-8(D6A) interfered with the binding of FSH to its receptor.