Background and Aims: Acute liver failure (ALF) is an inflam-matory process of acute liver cell injury. Mesenchymal stem cells (MSCs) are undifferentiated, primitive cells with anti-inflammatory, anti-apoptotic, and multi-directional differen-tiation abilities. This study aimed to explore the therapeutic mechanism of umbilical cord (U)MSCs in ALF. Methods: D-galactosamine (D-GalN) combined with lipopolysaccharide (LPS) was used to establish an ALF model. After model estab-lishment, UMSCs were injected via the tail vein. After UMSC transplantation, the number of mouse deaths was moni-tored every 12 h. A fully automatic biochemical analyzer was used to detect changes in biochemical analysis. Pathological changes was observed by stained with hematoxylin and eo-sin.The expression of My D88 was detected by immunohis-tochemical analysis, quantitative reverse transcription, and western blotting. The expression of NF-κB was detected by quantitative reverse transcription, western blotting.The ex-pression of Bcl-2,Bax were detected by quantitative reverse transcription, western blotting.The expression of TNF-α, IL-1β, IL-6 were detected by enzyme-linked immunosorbent assay. Results: The 48-h survival rate of the UMSC-treated group was significantly higher than that of the LPS/D-GalN-exposed group. After 24 h of LPS/D-GalN exposure, UMSCs reduced serum alanine aminotransferase and aspartate ami-notransferase levels and improved the liver structure. West-ern blot and real-time fluorescence quantitative nucleic acid amplification analyses showed that UMSCs decreased MyD88 expression, thereby inhibiting LPS/GalN-induced phosphoryl-ation and degradation of inhibitor of nuclear factor (NF)-κB (IκB). Additionally, NF-κB p65 underwent nuclear transloca-tion, inhibiting the production of the inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α and played a protective role in ALF by down-regulating the pro-apoptotic gene Bax and up-regulating the anti-apoptotic gene Bcl-2. In summary, these findings indicate that UMSCs play a protective role in LPS/GalN-induced acute liver injury via inhibition of the MyD88 pathway and subsequent inhibi-tion of NF-κB-mediated cytokine production. Conclusions: Through the above mechanisms, UMSCs can effectively re-duce LPS/D-GalN-induced ALF, reduce mouse mortality, and restore damaged liver function and damaged liver tissue.