Abstract: The transcription factors of NR1D1 and NR1D2 belong to the nuclear receptor transcription factor family. They participate in many biological processes by regulating the expression of target genes and play an important role in the regulation of circadian rhythm. In this study, we revealed the functional role of NR1D1 and NR1D2 underlying low-temperature environments in fish by analyzing their molecular characteristics and gene regulatory relationships in pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) under cold stress and their biological pathways involved. Bioinformatics comparative analysis results demonstrated that bothNR1D1 and NR1D2gene sequences had 23 open reading frames and the encoded proteins were unstable hydrophilic proteins. Both NR1D1 and NR1D2 proteins contained ZF-C4 type zinc lipoprotein domain and the ligand binding domain Hormone_Recep of nuclear hormone receptor, and the main ligand was Protoporphyrin Ix Containing Fe. Moreover, these two proteins mainly play the role in regulating gene expression in the nucleus and have direct regulations in many key genes of the circadian rhythm pathway. Results showed that NR1D1 and NR1D2, as transcription factors, could participate in the regulation of circadian rhythm and promote glucose and lipid metabolism, mitochondrial oxidation and other processes by regulating the expression of target genes under cold stress, providing necessary energy for pearl gentian grouper and helping the enhancement of cold tolerance.