Objective To investigate the regulatory effect of nuclear transcription factors thyroxine receptor (TRB) and retinoic acid receptor X (RXR), as well as the agonist T4, on Cdc42-interacting protein 4 ( Cip 4) gene expression. Methods The Cip 4 gene promoter of Mongolian gerbils was cloned into the luciferase-reporter sequence of pGL3 basic to construct a new plasmid pGL3- Cip 4. Coding sequences of transcription factors TRB and RXR were cloned into pcDNA3. 1 eukaryotic expression vectors. pGL3- Cip 4, pcDNA3. 1-TRB, pcDNA3. 1-RXR, and pRL-TK (reference plasmid) vectors were used to transfect HEK-293 cells to form a luciferase double-reporter system for the Cip 4 gene. Transfection efficiency was optimized by adjusting the ratio of these plasmids. The agonist T4 was used to analyze transcriptional activity. Results Transfection efficiency and luciferase activity were the highest when a total amount of 2 μg plasmid and a ratio of 20∶1 between the (promoter + transcription factor) and pRL-TK. RXR could significantly increase Cip 4 gene transcription of ( P < 0. 05), whereas TRB did not alter Cip 4 transcription ( P > 0. 05). Thyroxine T4 could enhance RXR upregulation ( P < 0. 001), while a combination of T4 and TRB downregulated Cip 4 transcription ( P < 0. 05). Levels of Cip 4 transcription were significantly downregulated in the presence of T4, RXR, and TRB ( P < 0. 01). Conclusions T4, TRB, and RXR had coregulatory effects on Cip 4 gene transcription in Mongolian gerbils; moreover, they simulated the process of ligand T4 agonizing TRB/ RXR to activate Cip 4 gene transcription. These result indicating that Cip 4 transcription was significantly affected by the RXR signaling pathway lay the foundation for revealing thyroxine’ s mechanism of action in human nonalcoholic fatty liver disease, as well as the study of ligand-agonizing nuclear transcription factor regulation of Cip 4 gene expression and related drug screening.