The Forkhead box (Fox) protein family is a family of transcription factors with wing-like helical structured DNA-binding region and is widely distributed in biological groups ranging from yeast to mammals. The Fox protein family is divided into 19 subfamilies, A to S, among which the forkhead box O (FoxO) subfamily is the most intensively studied. Forkhead box O4 (FoxO4) is a member of the FoxO subfamily, which includes FoxO1, FoxO3, and FoxO6 in mammals. These members share structural and functional similarities as well as regulations. In certain fish species, seven highly homologous members of the FoxO subfamily exist: FoxO1a, FoxO1b, FoxO3a, FoxO3b, FoxO4, FoxO6a, and Fox06b. The involvement of FoxO in the regulation of various physiological functions, including the cell cycle, apoptosis, DNA damage repair, oxidative stress, cell differentiation, and glucose metabolism, has been demonstrated in multiple studies. Furthermore, its activity is modulated through diverse mechanisms such as phosphorylation, acetylation, and ubiquitination. To explore the molecular characteristics of foxo4 in grass carp (Ctenopharyngodon idella) and its relationship with the nutritional regulation of dietary proteins, we obtained the grass carp foxo4 sequence through homologous cloning. Its open reading frame is 1875 bp, encoding 624 amino acids, and it consists of three functional domains: FH, Pfam (FOXO_KIX_bdg), and Pfam (FOXO_TAD). Amino acid sequence alignment analysis showed that the foxo4 gene in C. idella is highly homologous to that in Danio rerio, Homo sapiens, Mus musculus, and Electrophorus electricus. Analysis of codon usage bias revealed that foxo4 exhibited a strong preference for CUG and AGC codons across all examined species, whereas C. idella and Pimephales promelas shared certain similarities in their codon preferences, suggesting a close evolutionary relationship between them. Phylogenetic tree analysis showed that the foxo4 gene of grass carp has the highest homology to that of Pimephales promelas. Tissue expression analysis of foxo4 revealed that the mRNA expression level of foxo4 was most significant in grass carp muscle (P＜0.05), followed by the heart, liver, intestine, brain, spleen, and kidney. In addition, circadian rhythm analysis showed that the expression of foxo4 was the highest at 18:00 and lowest at 24:00. This study also explored the effects of dietary protein on the expression of foxo4. The results of different protein source experiments showed that after 14, 21, and 35 days of the feeding trial, the expression of the foxo4 gene in the intestinal tissue of grass carp was significantly upregulated in both the rapeseed meal and fish meal groups compared with that in the soybean meal group (P＜0.05). These findings suggest a close association between the expression of foxo4 gene in grass carp and dietary protein sources. A feeding trial with different levels of l-alanyl-l-glutamine dipeptide (Ala-Gln) showed a gradual decrease in the expression of foxo4 in the intestines of grass carp as the ratio of Ala-Gln in the feed increased. The control group showed the highest expression level, whereas the 1.5% addition group displayed the lowest expression level. In summary, the expression of foxo4 in grass carp exhibits tissue specificity and is regulated by dietary protein sources and dipeptide levels. This study establishes a foundation for revealing the molecular characteristics of foxo4 in fish and its protein nutritional response and provides basic data for subsequent studies on the molecular mechanism of foxo4 gene regulation in fish protein metabolism in teleosts.