Acute-on-chronic liver failure (ACLF) constitutes a prevalent manifestation of liver failure within clinical settings. This condition manifests swiftly and is characterized by an exceedingly elevated fatality rate.While numerous investigations have delved into the role of RNA methylation in ferroptosis, the impact of such methylation on ACLF-associated ferroptosis remains notably underexplored. This study aimed to elucidate the molecular mechanism underlying the efficacy of Niujiao Dihuang Jiedu decoction (NDD) in mitigating ferroptosis in ACLF, with a specific focus on RNA 5-methylcytosine (m5C) methylation.An ACLF rat model was established alongside an erastin-induced ferroptosis model in LO2 cells. Both in vitro and in vivo experiments were conducted to substantiate NDD's influence on ferroptosis. The modifying influence of methylase NOL1/NOP2/sun domain (NSUN5) upon SLC7A11, a key ferroptosis-associated gene, was probed through dot blot, immunofluorescence co-localization, and RNA binding protein immunoprecipitation (RIP) experiments.Serological and hepatic histopathological findings indicated NDD's discernible therapeutic impact on ACLF. Furthermore, ferroptosis phenotype experiments revealed NDD's proficiency in effectively impeding the occurrence and development of ferroptosis. Dot blot assays demonstrated a reduction in the overall RNA m5C levels during cellular ferroptosis. Furthermore, through immunofluorescence co-localization and RIP techniques, we found that the propensity of methylase NSUN5 to associate with SLC7A11 mRNA, thereby enhancing its protein translation and conferring resistance against ferroptosis.RNA methylation is involved in the process of ACLF-associated ferroptosis, and NDD can inhibit ACLF-associated ferroptosis by fostering SLC7A11 m5C methylation.Copyright © 2023. Published by Elsevier GmbH.