The structure and crystallographic orientation of different layers (the cone layer, palisade layer and the crystal layer) in ostrich eggshells were studied by scanning electron microscope (SEM). X-ray diffraction (XRD) and transmission electron microscope (TEM) with selected area electron diffraction (SAED). It is found that the degree of 0 0 1 preferred orientation enhanced from inner to outer ostrich eggshells. A crystallographic orientation regulation was observed for the first time. The adjacent 1-5 calcite crystals with the same three-dimensional orientation are proposed to form a crystalline cluster with the size of several microns. The a-axes in the neighboring clusters orient from several degrees to tens of degrees.