Three-dimensional (3D) reconstruction serves as a crucial instrument for the analysis of biological structures. In particular, a comprehensive and accurate 3D ultrastructural examination of rat sperm is vital for understanding and diagnosing male fertility issues and the underlying causes of infertility. In this study, we utilized the automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) imaging technique, a highly effective method for 3D cellular ultrastructural analysis. Our findings reveal that during spermiogenesis, the volume of the nucleus undergoes a significant reduction, shrinking to just 10% of its original size. The acrosomal vesicles, derived from the Golgi apparatus, converge and elongate along the spermatid nucleus. These vesicles then attach to the nucleus via a cap-like structure, thereby defining the head side of the spermatozoa. In the initial stages of spermiogenesis, the mitochondria in spermatids are distributed beneath the cell membrane. As the process progresses, these mitochondria gradually migrate to the sperm tail, where they form the mitochondrial sheath. This sheath plays a crucial role in providing the energy required for the movement of the sperm. In addition, we also reconstructed the mRNA-stroring sturcture-chromatoid body in sperm cells, which were cloud-like or net-like structures in the cytoplasm. The precise and comprehensive nature of 3D ultrastructural examination allows for a deeper understanding of the morphological process of spermiogenesis, thereby contributing to our knowledge of male fertility and the causes of infertility. Our research has significantly advanced the understanding of the 3D ultrastructure of sperm, more comprehensively than ever before.