Abstract: Nuclear erythroid 2 related factor 1 (nrf1), a transmembrane transcription factor, plays an important role in anti-oxidative stress in animals. In this study, the nrf1 gene sequence of grass carp (Ctenopharyngodon idella) was obtained by homologous cloning, with an open reading frame (ORF) spanning 1560 bp which encoded a polypeptide consisting of 519 amino acids. Phylogenetic tree analysis showed that nrf1 was closely related to Megalobrama amblycephala. The tissue expression analysis of nrf1 revealed the liver as the primary site of nrf1 expression, followed by the heart and intestine. Circadian rhythm analysis showed that the expression level of nrf1 was the highest at 9:00 and significantly higher than that at 3:00, 12:00, 18:00, 21:00 and 24:00 (P<0.05). After 24h and 48h of acute ammonia stress treatment, the expression of nrf1 gene was significantly upregulated in both low ammonia nitrogen (5 mg/L) and high ammonia nitrogen (20 mg/L) groups compared to the control group (0) (P<0.05). The expression level of nrf1 was significantly lower in the low ammonia nitrogen group compared to the high ammonia nitrogen group at 24h (P<0.05), and significantly higher than that in the high ammonia nitrogen group at 48h (P<0.05). Furthermore, growth experiments on grass carp were conducted using three distinct protein sources (fish meal, rapeseed meal, and soybean meal), revealing that the expression level of nrf1 was significantly higher in the rapeseed meal and soybean meal groups compared to the fish meal group at 14d, 28d, and 35d after feeding trial (P<0.05). At 28d, the expression level of nrf1 in the soybean meal group was notably higher than that in the rapeseed meal group. In conclusion, the expression of the nrf1 gene in grass carp exhibits tissue specificity and is regulated by ammonia nitrogen concentration in water as well as dietary protein source. This study provides a theoretical framework for elucidating the molecular characteristics of the nrf1 gene in fish and its pivotal role in mediating antioxidative stress response.