FK866 exhibits a protective effect on D-galactosamine (GaIN)/lipopolysaccharide (LPS) and concanavalin A (ConA)-induced acute liver failure (ALF), but the mechanism by which FK866 affords this benefit has not yet been elucidated. Autophagy has a protective effect on acute liver injury. However, the contribution of autophagy to FK866-conferred hepatoprotection is still unclear. This study aimed to investigate whether FK866 could attenuate GaIN/LPS and ConA-induced ALF through c-jun-N-terminal kinase (JNK)-dependent autophagy. In vivo, Mice were pretreated with FK866 at 24, 12, and 0.5 h before treatment with GaIN/LPS and ConA. 3-methyladenine (3MA) or rapamycin were used to determine the role of autophagy in FK866-conferred hepatoprotection. In primary hepatocytes, autophagy was inhibited by 3MA or autophagy-related protein 7 (Atg7) small interfering RNA (siRNA). JNK was suppressed by SP600125 or Jnk siRNA. FK866 alleviated hepatotoxicity and increased autophagy while decreased JNK activation. Suppression of autophagy abolished the FK866-conferred protection. Inhibition of JNK increased autophagy and exhibited strongly protective effect. Collectively, FK866 could ameliorate GaIN/LPS and ConA-induced ALF through induction of autophagy while suppression of JNK. These findings suggest that FK866 acts as a simple and applicable preconditioning intervention to protect against ALF; autophagy and JNK may also provide therapeutic targets for ALF treatment.