X-ray microtomography is rapidly gaining importance as a non-destructive investigation technique, especially in the three-dimensional examination of trabecular bone. Appropriate quantitative three-dimensional parameters describing the investigated structure were introduced, such as the model-independent thickness and the structure model index. The first parameter calculates a volume-based thickness of the structure in three dimensions independent of an assumed structure type. The second parameter estimates the characteristic form of which the structure is composed, i.e. whether it is more plate-like, rod-like or even sphere-like. These parameters are now experiencing a great diffusion and are rapidly growing in importance. To measure the accuracy of these three-dimensional parameters, a physical three-dimensional phantom containing different known geometries and thicknesses, resembling those of the examined structures, is needed. Unfortunately, such particular phantoms are not commonly available and neither does a consolidated standard exist. This work describes the realization of a calibration phantom for three-dimensional X-ray microtomography examination and reports an application example using an X-ray microtomography system. The calibration phantom (external size 13 mm diameter, 23 mm height) was based on various aluminium inserts embedded in a cylinder of polymethylmethacrylate. The inserts had known geometries (wires, foils, meshes and spheres) and thicknesses (ranging from 20 μm to 1 mm). The phantom was successfully applied to an X-ray microtomography device, providing imaging of the inserted structures and calculation of three-dimensional parameters such as the model-independent thickness and the structure model index. With the indications given in the present work it is possible to design a similar phantom in a histology laboratory and to adapt it to the requested applications.