AbstractBackgroundAcute liver failure (ALF) is a serious liver disease that is difficult to treat owing to its unclear pathogenesis. This study aimed to investigate the roles and molecular mechanisms of calycosin (CA) in ALF.MethodsIn this study, the roles and mechanism of CA in ALF were explored using an in vitro lipopolysaccharide (LPS)‐induced ALF cell model. Additionally, 3‐(4,5‐dimethyl‐2‐thiazolyl)−2,5‐diphenyltetrazolium bromide assay was used to assess the effect of CA on the activity of LPS‐induced L02 human liver epithelial cells, and flow cytometry was used to detect apoptosis in L02 cells. Expression levels of apoptosis‐related genes,BaxandBcl‐2, were measured using reverse transcription‐quantitative polymerase chain reaction and Western blot analysis. Expression levels of inflammatory factors in LPS‐induced L02 cells were measured using an enzyme‐linked immunosorbent assay. Additionally, the effect of CA on ALF was inhibited via transfection of a toll‐like receptor 4 (TLR4)‐plasmid to elucidate the relationship between CA and TLR4/nuclear factor (NF)‐κB signaling pathway in ALF.ResultsCA had no toxic effects on L02 cells, but enhanced the activity of LPS‐induced L02 cells in a dose‐dependent manner. Apoptosis and inflammatory factor release was increased in ALF, activating theTLR4/NF‐κBsignaling pathway. However, CA treatment inhibited the apoptosis and release of inflammatory factors. Further mechanistic studies revealed that the upregulation ofTLR4expression reversed the alleviating effects of CA on inflammation and apoptosis in LPS‐induced L02 cells.ConclusionCA alleviates inflammatory damage in LPS‐induced L02 cells by inhibiting theTLR4/NF‐κBpathway and may be a promising therapeutic agent for ALF treatment.