Prostate cancer is the most frequently diagnosed cancer and the second major cause of cancer related death in men of the Western society. The lack of effective treatments for advanced stages of prostate cancer demands a need to develop new strategies such as gene therapy that can be use either separately or in conjunction with current treatment methods. Despite significant advances in cancer gene therapy within the past decade, progress to clinical settings has been slowed due to several technical challenges. Gene delivery based on Adenoviral (Ad) vectors has considerable potential for treatment of cancer. However, the elicitation of host immune response by Ad-based vectors can lead to systemic toxicity and limit the duration of transgene expression consequently hinder the application of these vectors for human gene therapy. Hence, methods designed to elude immunogenicity of Ad vectors would be extremely advantages. Further enhancement in safety and efficacy requires selective gene expression and appropriate route of vector delivery. Finally, the capability to non-invasively monitor transgene expression, viral biodistribution and treatment outcome would advance clinical gene therapy trials. The goal of this dissertation is to address these challenges toward achieving safer and more effective Ad-based gene therapy for prostate cancer.