Chronic and excessive alcohol consumption leads to alcoholic liver disease (ALD). However, the molecular mechanisms in the regulation of ALD have not been fully deciphered. Liver lipid accumulation is an important research direction in ALD. In this study, the physiological role of nuclear factor Y (NF-Y) in ALD and the related mechanisms were investigated using murine hepatocytes and an ethanol-induced liver injury mouse model. In this study, ethanol promoted hepatic NF-Y expression in a mouse model and Hepa1-6 mouse hepatocytes. Lentivirus-mediated NF-Y overexpression in Hepa1-6 cells markedly increased sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) expression compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated FASN and SREBP1 expression. Mechanistically, luciferase reporter gene assays and chromatin immunoprecipitation (ChIP) analysis indicated that NF-Y activates the transcription of SREBP1 by directly binding to the CCAAT regulatory sequence motif in the promoter. Overall, our results reveal a previously unrecognized physiological function of NF-Y in ALD by activating sterol regulatory element-binding protein 1 (SREBP1). Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage ALD. (C) 2021 Published by Elsevier Inc.