Abstract We have developed a 3D-imaging system using characteristic X-rays produced by proton micro-beam bombardment. The 3D-imaging system consists of a micro-beam and an X-ray CCD camera of 1 mega pixels (Hamamatsu photonics C8800X), and has a spatial resolution of 4 μm by using characteristic Ti–K-X-rays (4.558 keV) produced by 3 MeV protons of beam spot size of ∼1 μm. We applied this system, namely, a micron-CT to observe the inside of a living small ant's head of ∼1 mm diameter. An ant was inserted into a small polyimide tube the inside diameter and the wall thickness of which are 1000 and 25 μm, respectively, and scanned by the micron-CT. Three dimensional images of the ant's heads were obtained with a spatial resolution of 4 μm. It was found that, in accordance with the strong dependence on atomic number of photo ionization cross-sections, the mandibular gland of ant contains heavier elements, and moreover, the CT-image of living ant anaesthetized by chloroform is quite different from that of a dead ant dipped in formalin.