Background: The poultry industry experiences genetic losses due to recurring infectious diseases, necessitating effective preservation strategies. Nitric oxide plays a crucial role in male reproduction, and optimal NO (nitric oxide) levels may enhance sperm viability. This study investigated the effects of SNAP (S-nitroso-Nacetylpenicillamine) on the longevity of rooster sperm. Methods: Semen was diluted with Beltsville Poultry Semen Extender-I containing 0 or 25 μM SNAP and stored at 10°C. Sperm motility and acrosome integrity were assessed at 1, 3, and 7 days. NO levels were quantified by DAF-FM diacetate and AI trials were evaluated by fertility and hatchability. Results: On day 1, sperm motility in the SNAP 25 μM-treated group was significantly higher than in the control. NO quantification confirmed that SNAP-treated semen exhibited higher NO levels. For fertilization and hatchability assessment, hens were divided into two groups based on the presumed duration sperm resided in sperm storage tubules. Before artificial insemination, the sperm was preserved at low temperature (10°C) to maintain viability. Fertilization rates were significantly higher in the SNAP-treated group in both short-term and long-term SST storage conditions. However, hatchability was only significantly improved in the SNAP-treated group when fertilization occurred after extended storage. Conclusions: These findings suggest that NO enhances sperm viability and fertility in poultry semen stored at low temperatures. SNAP 25 μM enhances AI efficiency by maintaining sperm viability and extending fertilization potential. Further research is needed to refine NO-based fertility enhancement strategies for avian species.