Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. Invitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for invivo analysis to quantify Ad infection within the tumor. Invitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p=0.80). Invivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1×106 plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p=0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues. © 2013 World Federation for Ultrasound in Medicine & Biology.