Prostate cancer (PCa) is the second leading cause of cancer-related deaths in Western male population. Previous studies have demonstrated that differential display code 3 (DD3 or DD3(PCA3)) is one of the most PCa-specific genes; therefore, it has been used as a clinical diagnostic marker for PCa. In this study, we constructed an oncolytic adenovirus Ad.DD3-E1A by using the minimal DD3 promoter to replace the native viral promoter of E1A gene. In addition, Ad.DD3-E1A was armed with therapeutic gene IL-24 to enhance its antitumor activity. The resulting adenovirus, Ad.DD3-E1A-IL-24, demonstrated PCa specificity and excellent antitumor effect. Further analyses on its antitumor mechanism revealed that it has the capacity to induce apoptosis in PCa cells and inhibit angiogenesis. These results suggest that Ad.DD3-E1A-IL-24 is a promising antitumor agent that may be able to be used in the future as a treatment for PCa.