Recently, electro-gene transfer has become a powerful tool to enhance the efficiency of gene expression in different organs and particularly in muscle, which provides efficient secretion of therapeutic proteins into the circulation. However, its toxicity, owing to the high field strengths of conventional direct current (DC) square-waves, should be taken into account and should be minimized if electroporation is to be used routinely for the treatment of human diseases. In this study, we demonstrate that efficient in vivo gene transfer could be safely achieved using pulses of alternating current sine-waves (ACSWs) with a frequency of 60 Hz. Importantly, the field strength was decreased to as low as 20 V/cm and the efficiency of muscle gene transfer increased more than tenfold and showed less toxicity than with conventional DC square-wave pulses. Using ACSW pulses to transfer human clotting factor IX (hFIX) plasmid into muscle, we observed significant phenotypic correction in mice with hemophilia B.