The feeding of high-energy and low-protein diets often induces fatty liver hemorrhagic syndrome (FLHS) in laying hens. However, the mechanism of hepatic fat accumulation in hens with FLHS remains uncertain. In this research, a comprehensive hepatic proteome and acetyl-proteome analysis was performed in both normal and FLHS-affected hens. The results indicated that the upregulated proteins were primarily associated with fat digestion and absorption, the biosynthesis of unsaturated fatty acids, and glycerophospholipid metabolism, while the downregulated proteins were mainly related to bile secretion and amino acid metabolism. Furthermore, the significant acetylated proteins were largely involved in ribosome and fatty acid degradation, and the PPAR signaling pathway, while the significant deacetylated proteins were related to valine, leucine, and isoleucine degradation in laying hens with FLHS. Overall, these results demonstrate that acetylation inhibits hepatic fatty acid oxidation and transport in hens with FLHS, and mainly exerts its effects by affecting protein activity rather than expression. This study provides new nutritional regulation options to alleviate FLHS in laying hens.