Sarco/endoplasmic reticulum Ca²+-ATPase (SERCA2a) is important for cardiac physiological function and pathological progression. However, intravenous injection, a commonly applied approach for gene delivery in most studies investigating the expression of SERCA2a in cardiomyocytes, has not been particularly satisfactory. Therefore, in the present study, a modified method was used to transfect this gene into the heart. Specifically, a SERCA2a-knockdown lentivirus was directly injected into the myocardium of adult rats under ultrasound guidance, following which the effectiveness and feasibility of this proposed approach were evaluated. The results demonstrated that compared with traditional intravenous injection, the modified gene delivery method resulted in markedly higher transfection efficiency. In addition, the SERCA2a-knockdown rats exhibited higher rates of arrhythmia and weaker ventricular wall motions compared with those in the control rats, with these symptoms more evident in the rats that received a direct injection into the myocardium compared with those that were intravenously injected. These results suggest that ultrasound-guided injection into the myocardium is an efficient and safe method for gene delivery and for inducing the knockdown of SERCA2a protein expression in cardiomyocytes in their native environment.