Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured invitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3h invitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured invitro.