Although micro-computed tomography (micro-CT) images have high contrast for bone or air, between soft tissues the contrast is typically low. To overcome this inherent issue, attenuating exogenous contrast agents are used to provide contrast enhancement in the vasculature and abdominal organs. The aim of this study is to measure the contrast enhancement time course for a gold nanoparticle blood-pool contrast agent and use it to perform cardiac-gated 4D micro-CT scans of the heart. Six healthy female C57BL/6 mice were anesthetized and imaged after receiving an injected dose of MVivo gold nanoparticle blood-pool contrast agent. Following the injection, we performed micro-CT scans at 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 24, 48 and 72 h. The mean CT number was measured for 7 different organs. No contrast enhancement was noticed in the bladder, kidneys or muscle during the time-course study. However, it clearly appears that the contrast enhancement is high in both right ventricle and vena cava. To perform cardiac-gated imaging, either the gold nanoparticle agent (n = 3) or an iodine-based (n = 3) contrast agent was introduced and images representing 9 phases of the cardiac cycle were obtained in 6 additional mice. A few typical cardiac parameters were measured or calculated, with similar accuracy between the gold and iodinated agents, but better visualization of structures with the gold agent. The MVivo Au contrast agent can be used for investigations of cardiac or vascular disease with a single bolus injection, with an optimal cardiac imaging window identified during the first hour after injection, demonstrating similar image quality to iodinated contrast agents and excellent measurement accuracy. Furthermore, the long-lasting contrast enhancement of up to 8 h can be very useful for scanning protocols that require longer acquisition times.