Hibernation is an adaptive survival strategy in response to cold and foodless winter. To determine the underlying mechanisms of seasonal adaptions, transcriptome sequencing studies have been conducted in bears, ground squirrels and bats. Despite advances in identifying differentially expressed genes involved in metabolism, the precise mechanisms of these physiological adaptions remain unclear. In the present study, we examined liver of Chinese Soft-Shelled Turtle (Pelodiscus sinensis) and found that the contents of lipid droplet (LD) and triglyceride (TG) were significantly decreased during hibernation. Increases in mRNA expression levels of lipolysis-related genes and decreased levels of lipogenesis-related genes during hibernation indicated that LD hydrolysis was stimulated during hibernation. To continuously release fatty acids (FAs) from LD, adipose triglyceride lipase (ATGL) was recruited and accumulated on the surface of LDs via activation of Cyclic Adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling. Meanwhile, increased phosphorylation of the LD-associated protein, perilipin-5, activated the enzyme activity of ATGL via interaction between comparative gene identification-58 (CGI-58) and ATGL. Taken together, our results indicated that ATGL accumulation on the LD surface and its induced enzyme activity during hibernation promoted LD breakdown in the liver of Chinese Soft-Shelled Turtle (Pelodiscus sinensis), thereby enhancing mitochondrial β-oxidation to maintain energy hemostasis.