<span class="paragraphSection"><div class="boxTitle">Abstract</div>The sterol regulatory element-binding transcription factor 1 (SREBP-1) plays a crucial role in the transcriptional regulation of lipogenic response genes, thereby contributing to the development of non-alcoholic fatty liver disease (NAFLD). However, the modulation of SREBP-1 transcriptional activity remains incompletely understood. Here, we report that the transcription factor FOXN3 interacts with the KU70/KU80/SREBP-1 complex, facilitating the recruitment of SREBP-1 for the transcriptional activation of lipogenic response genes. Hepatocyte-specific knockout of FOXN3 significantly alleviates the pathological progression of NAFLD by suppressing fatty acid and cholesterol synthesis. Furthermore, phosphorylation of FOXN3 at the S83 and S85 residues disrupts the stability of the KU70/KU80/FOXN3/SREBP-1 complex, which is required for SREBP-1 transcriptional activity. This disruption consequently impedes the progression of NAFLD. Clinical investigations reveal that FOXN3, KU80, and SREBP-1 co-target the promoters of lipogenic response genes in fatty liver tissues from patients. Notably, phosphorylation levels of FOXN3 at S83 and S85 are significantly reduced in fatty liver tissues compared to normal samples. This reduction enhances the enrichment of the FOXN3/SREBP-1 complex at the promoters of lipogenic response genes during the progression of NAFLD. Our study underscores the critical role of FOXN3 in maintaining the intact KU70/KU80/FOXN3/SREBP-1 complex, which is essential for SREBP-1-mediated metabolic disorders.</span>