Objective To assess the efficiency and toxicity of adeno-associated virus serotype 9 (AAV9) mediated sarcoplasmic reticulum Ca2+ATPase 2a (SERCA2a) gene transferring to myocardium in SD rats by different cardiac gene delivery methods. Methods The AAV9-SERCA2a-EGFP virus victor system was constructed in vitro successfully. According to the three cardiac gene delivery methods, 90 normal SD rats were randomly divided into three groups: TVI (Tail vein injection，TVI)group, IMI（Intramyocardial injection，IMI）group, and IPI (Intrapericardial injection，IPI)group. Each group was respectively transferred with AAV9-SERCA2a-EGFP, AAV9-EGFP, NS 200µL at the titer of 1×1011 vg/mL using the appropriate method. After 30 days of gene delivery, the expression of EGFP in the tissue of heart, liver and kidney was observed by inverted fluorescence microscopy. Western blotting was performed to detect relative expression levels of SERCA2a gene in rats' tissue. Surface 12-lead ECG was used to record the incidence of arrhythmia. HE staining observed the histopathological changes. The changes of cardiac function was measured by echocardiogram. Blood chemistry indicators were used to assess the changes in liver and kidney. Results Left ventricular could observed a large number of green fluorescent in three groups and IMI group of green fluorescent confined in the injection point, liver and kidney were seen faint green fluorescence in three groups; The relative expression of SERCA2a protein of myocardium in the three groups was significantly higher than liver and kidney (P<0.01), and the myocardial relative expression of SERCA2a protein in TVI group and IMI group was significantly higher than IPI group (P<0.01). ECG was normal in TVI group and IPI group, but frequent premature ventricular beats were observed in two rats of IMI group.There were no significant difference in cardiac function, histopathology and blood chemistry indicators between transferring AAV9-SERCA2a-EGFP, AAV9-EGFP and transferring NS (P>0.05). Conclusion Transfected with a titer of 1×1011 vg/mL AAV9-SERCA2a gene, the efficiency in TVI group and IMI goup was better than IPI goup, and the toxicity in TVI group and IPI goup was lower than IMI group. Considering the evaluation of efficiency and toxicity by 1×1011 vg/mL AAV9-SERCA2a gene delivering in three methods, the TVI method is better than IMI and IPI method.