Acute liver failure (ALF) is a severe life-threatening disease associated with the disorder of the gut-liver axis. However, the cellular characteristics of ALF in the gut and related therapeutic targets remain unexplored. Here, we utilized the D-GALN/LPS (D/L)-induced ALF model to characterize 33,216 single-cell transcriptomes and define a mouse ALF intestinal cellular atlas. We found that unique, previously uncharacterized intestinal immune cells, including T cells, B cells, macrophages, and neutrophils, are responsive to ALF, and we identified the transcriptional profiles of these subsets during ALF. We also delineated the heterogeneity of intestinal epithelial cells (IECs) and found that ALF-induced cell cycle arrest in intestinal stem cells and activated specific enterocyte and goblet cell clusters. Notably, the most significantly altered IECs, including enterocytes, intestinal stem cells and goblet cells, had similar activation patterns closely associated with inflammation from intestinal immune activation. Furthermore, our results unveiled a common Ep300-dependent transcriptional program that coordinates IEC activation during ALF, which was confirmed to be universal in different ALF models. Pharmacological inhibition of Ep300 with an inhibitor (SGC-CBP30) inhibited this cell-specific program, confirming that Ep300 is an effective target for alleviating ALF. Mechanistically, Ep300 inhibition restrained inflammation and oxidative stress in the dysregulated cluster of IECs through the P38-JNK pathway and corrected intestinal ecology by regulating intestinal microbial composition and metabolism, thereby protecting IECs and attenuating ALF. These findings confirm that Ep300 is a novel therapeutic target in ALF and pave the way for future pathophysiological studies on ALF.