Cardiovascular gene therapy aims to treat coronary and peripheral artery disease, heart failure, and arrhythmia. The chosen transgene, delivery method, gene therapy vector type, high-quality vector production, and dose are all determining factors of the therapeutic outcome. High-resolution vascular imaging and increased knowledge of vascular biology in physiological and pathological conditions enable the finding of novel molecular targets for cardiovascular gene therapy. Transgenic and knockout mouse models have provided researchers several powerful experimental tools for studying the effects of single genes on cardiovascular diseases. For preclinical efficacy, safety, and toxicology studies, large animal models are needed before entering into clinical testing. This review focuses on commonly used animal models in cardiovascular gene therapy and describes recent advancements in the field of vascular biology. Emphasis is also given on high-resolution imaging of microvasculature and its impact on our knowledge of vascular function.