Objective To establish a cardiomyocyte model over-expressing mutant human PRKAG2 by infecting neonatal SD rat myocardial cells with constructed recombinant adenovirus vector Ad-PRKAG2 (R302Q)-IRES2-EGFP. Methods PRKAG2 (R302Q)-IRES2-EGFP was directly cloned into entry vector pDONR221 by using Invitrogen Gateway^TM technology. Then BP and LR recombination reactions yielded the recombinant adenovirus vector containing human PRKAG2 (R302Q) gene. The pAd-PRKAG2 (R302Q)-IRES2-EGFP was digested by Pac Ⅰ, and transfected into 293 cells. After packaging, amplification and purification, the virus was used to infect neonatal rat cardiomyocytes. Then the expression of PRKAG2 protein was assayed by Western blotting analysis in the infected neonatal SD rat cardiomyocytes. Results Restriction enzyme digestion analysis and the sequence analysis confirmed that PRKAG2(R302Q) gene was successfully inserted into the adenovirus vector. The myocardial cells infected with Ad-PRKAG2(R302Q)-IRES2-EGFP gave off strikingly bright green fluorescence and PRKAG2 protein was proven significantly over-expressed by Western blotting analysis (P<0.05). Conclusion The recombinant adenovirus containing human PRKAG2(R302Q) gene has been successfully constructed and expressed in neonatal rat cardiomyocytes, which paves a way for further study of PRKAG2 (R302Q) gene mutation.