Objective To explore the effects of hepatocyte-specific overexpression of adenosine kinase (ADK) on glucose intolerance and hepatic steatosis induced by high fat diet in mice. Methods Twenty C57BL/6J male mice were randomly divided into two groups (n=10):the mice in one group were injected through tail vein with adeno-associated virus vector carrying thyroxine-binding globulin (TBG) promotor driven expression of ADK (AAV8-TBG-ADK group), while those in the other group were injected with control vector AAV8-TBG (AAV8-TBG group). After adeno-associated virus administration, the mice in the two groups were randomly divided into two subgroups (n=5) to receive normal or high fat diet for the following 8 weeks, and changes in mice body weight were recorded. Mice were sacrificed after modeling. The expressions of ADK in the liver were determined by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The glucose homeostasis was assessed by glucose tolerance test. The pathological changes and lipid deposition of liver tissues were detected by Hematoxylin-Eosin (H-E) staining and oil red O staining, respectively. The mRNA levels of gluconeogenesis related genes (glucose-6-phosphatase [G6P] and phosphoenolpyruvate carboxykinase [PEPCK]) and fatty acid synthesis related genes (sterol-regulatory element binding protein-1 [SREBP-1], acetyl-CoA carboxylase-1 [ACC-1], fatty acid synthase [FAS] and stearoyl-CoA desaturase-1 [SCD-1]) in liver tissues were analyzed by qRT-PCR. Results Compared with the normal diet subgroup, high fat diet induced weight gain, glucose intolerance and hepatic steatosis. There was no significant difference in the body weight between the AAV8-TBG and AAV8-TBG-ADK groups (P>0.05). Compared with the AAV8-TBG group, AAV-TBG-ADK mediated ADK overexpression significantly ameliorated high fat diet-induced glucose intolerance and hepatic steatosis (all P<0.05), and significantly reduced the mRNA levels of gluconeogenesis related genes and fatty acid synthesis related genes in liver tissues (all P<0.05). Conclusion Hepatocyte-specific overexpression of ADK can improve high fat diet-induced glucose intolerance and hepatic steatosis in mice.