Aim To investigate the influence of nitric oxide (NO), nitric oxide synthase (NOS) on cardiomyocyte hypoxia/reoxygenation and effects of NO on cardiomyocytes delayed protection (DP). Methods The content of NO, NOS were measured in the models of hypoxia/reoxygenation (H/R) of cultured neonatal rat cardiomyocytes. The cell viability, lactate dehydrogenase (LDH) release, and the content of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured in the models of DP of cultured neonatal rat cardiomyocytes, with pretreatment of L-NA, L-arginine, sodium nitroprusside (SNP). Results NO and NOS increased significantly after transient hypoxia/reoxygenation. Hypoxic DP attenuated cardiomyocyte injure induced by H/R. Pretreatment with non-selective NOS inhibitor L-NA abolished the protectiive effect of hypoxia DP, L-arginine can not mimicked DP, but SNP may mimicked DP. Conclusion NO may be involved in the protection mechanism of DP.