The interest in using small animal models of human disease has produced a need to design a CT system at a microscopic level comparable to that achievable in a clinical CT in human. In this study, we developed a high-resolution in vivo-CT system with respiratory and cardiac gating using synchrotron radiation. The system was constructed in BL20B2 at SPring-8. SPring-8 is the third generation synchrotron radiation source in Hyogo, Japan, and prefers much higher flux X-ray than a laboratory X-ray source. Another advantage of synchrotron monochromatic CT is the minimalization of beam hardening effects, which pose serious problems when using white X-rays. Since the X-ray beam from the synchrotron source is parallel, each horizontal line corresponds to a slice position along the rotation axis. Multiple slices are easily obtained in one rotation (3D-CT). For in vivo scanning, the X-ray mechanical shutter and CCD electrical shutter were synchronized with an airway pressure (respiratory) and electrocardiographic (ECG) signal. Synchronization reduced the motion artifacts caused by respiration and heart beats, markedly improving visualization of the edges of the heart, ribs and diaphragm. In particular, small airways (diameter > 300 μm) and cerebral blood vessels were visualized clearly. This system is very useful for evaluating lung physiology and cardiovascular mechanics in vivo.