Lipid droplets (LDs) are intracellular organelles that store neutral lipids as energy reservoir. Recent studies suggest that autophagy is important in maintaining the homeostasis of intracellular LDs by either regulating the biogenesis of LDs, mobilization of fatty acids, or degradation of LDs in cultured cells. Increasing evidence also supports a role of autophagy in regulating glucose and lipid metabolism in vivo in mammals. In response to fasting/starvation, lipids are mobilized from the adipose tissue to the liver, which increases the number of intracellular LDs and stimulates fatty acid oxidation and ketogenesis. However, it is still controversial and unclear how impaired autophagy in hepatocytes affects the biogenesis of LDs in mouse livers. In the present study, it was demonstrated that hepatic autophagy-deficient (L-Atg)5 knockout mice had impaired adaptation to fasting-induced hepatic biogenesis of LDs. The maladaptation to fasting-induced hepatic biogenesis of LDs in L-Atg5 knockout mouse livers was not due to hepatic changes of de novo lipogenesis, secretion of very-low-density lipoprotein or fatty acid β-oxidation, but it was due to persistent nuclear factor-like 2 activation because biogenesis of LDs restored in L-Atg5/nuclear factor-like 2 double-knockout mice.