Objective To observe the effects of Lim protein Ajuba on transcriptional levels of key genes relevant to gluconeogenesis and explore the molecular mechanism of regulating the liver glycogen metabolism by Ajuba. Methods ①Human hepatoma cells HepG2 were infected with lentivirus and stable transfected cells with low/high expression of Ajuba were screened. The expression of Ajuba was detected by the Western blotting. ②Expressions of two key enzymes relevant to gluconeogenesis, i.e. phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P), were detected by the real-time PCR. ③Primary hepatocytes of C57BL/6 mice were isolated and cells with low expression of Ajuba were screened by the lentivirus infection method. The mRNA expressions of Pepck and G6p were detected by the real-time PCR. Results ①The results of Western blotting showed that HepG2 cells with low and high expressions of Ajuba were successfully screened. ②Compared with controls, mRNA levels of PEPCK and G6P in HepG2 cells with low expression of Ajuba significantly decreased, while mRNA levels of PEPCK and G6P in HepG2 cells with high expression of Ajuba significantly increased (P<0.05). ③The mRNA levels of Pepck and G6p in mouse primary hepatocytes with low expression of Ajuba significantly decreased (P<0.05). Conclusion The Lim protein Ajuba can participate in the regulation of glucose metabolism by up-regulating the expressions of PEPCK and G6P, two key genes relevant to gluconeogenesis. This effect is not species-specific.