As the understanding of human cancer biology increases, new potential strategies for gene therapy are being proposed and evaluated. However, safe and efficient gene transfer continues to be the major hurdle for its implementation in the clinic. Preclinical studies have shown how pulsed-high intensity focused ultrasound (HIFU) exposures can be combined with different modes of administration (local, intravascular and systemic) to improve local delivery of genes and other therapeutic agents. Using image guidance, exposures are given, where short pulses of energy create predominantly mechanical/structural effects in the tissues as opposed to thermal ones. The result is an increase in both extravasation and interstitial diffusion of macromolecules, which occur non-destructively and reversibly. Ultrasound contrast agents can also be added, which enhance acoustic cavitation activity and consequently sonoporation. By being able to locally increase the uptake and expression of DNA, pulsed-HIFU holds much promise to further the use and applications of gene therapy for treating cancer and other pathological conditions.