Almost all patients with advanced prostate cancer progress to castration-resistant stage with limited treatment options. Oncolytic adenoviruses have been actively pursued as potential agents for cancer treatment. Virtually all clinical trials on oncolytic adenovirus are based on serotype 5. However, viral replication in hepatocytes induces severe liver toxicity and limits its systemic administration for metastatic disease. Moreover, rapid clearance of viral particles injected intravenously further hinders the anticancer efficacy. Adenovirus 6 (Ad6) was previously reported to exhibit less liver toxicity and escape Kupffer cells absorption after systemic administration. To further improve its safety, we generated a novel oncolytic adenovirus Ad6miR, in which four copies of binding sites of a liver-specific microRNA miR122 were incorporated into E1A gene of Ad6. miR122 regulation significantly decreased Ad6 replication in hepatocytes and consequently hepatotoxicity because of the negative regulation of miR122. Cytotoxicity assay using primary or established prostate cancer cell lines showed robust oncolytic activity of Ad6miR. Systemic treatment of established tumors with Ad6miR showed strong antitumor activity, comparable with that of Ad6 or Ad5. Although Ad6 evaded Kupffer cells, its blood clearance rate was as rapid as Ad5. The vast majority of Ad6 particles intravenously injected localized in liver sinusoidal endothelial cells rather than previously reported Kupffer cells. Elevating Ad6miR injection dose increased circulating Ad6miR concentration and its antitumor efficacy. miR122 regulation of Ad6 significantly improves its safety profile after systemic administration, which allows increasing therapeutic doses leading to improved anticancer efficacy of systemic treatment of castration-resistant prostate cancer. Mol Cancer Ther; 11(11); 2410-8. (C) 2012 AACR.