Kaempferol, a flavonoid compound, has various biological functions. Acute liver failure (ALF) is a lethal clinical syndrome with severe liver function damage. In this study, we explored the mechanisms underlying the therapeutic effect of kaempferol in ALF. The ALF mouse model was established using D-galactosamine (D-GalN, 700 mg/kg)/lipopolysaccharide (LPS, 10 μg/kg). Two hours before the administration of D-GalN/LPS, different group of mice were pretreated according different doses of kaempferol, 6 hours after injection of D-GalN/LPS, and then killed. The survival rate, liver function and inflammatory cytokine levels were assessed. It was determined whether kaempferol pretreatment protected hepatocytes from ALF induced by D-GalN/LPS via autophagy pathway in vivo and in vitro. Pretreatment with a high dose of kaempferol significantly decreased the survival rate and increased severe liver damage; however, pretreatment with a low dose of kaempferol showed the opposite effect. Furthermore, pretreatment with a high dose of kaempferol augment the levels of proinflammatory cytokines and markers of the MAPK signaling pathway, while pretreatment with a low dose of kaempferol showed the opposite effect. Additionally, pretreatment with a high dose of kaempferol decreased autophagy, but pretreatment with a low dose of kaempferol increased autophagy in vivo and in vitro. It was also proved that pretreatment with 3-methyadenine (3- MA) or Atg7 siRNA to inhibit autophagy partially negated the hepatoprotective effect of kaempferol (5 mg/kg) pretreatment in ALF mice induced by D-GalN/LPS. Our findings demonstrate that effects of different doses of kaempferol on D-GalN/LPS-induced ALF is remarkably different by regulating the autophagy pathway.