The purpose of this study was to show the benefit of visualizing a tree-dimensional (3D) image of the tongue’s muscle structure, which until now has been regarded as fully understood. Until now, no suitable 3D observation methods have been developed for soft tissue, such as the tongue, using histological magnification. For this purpose, this study used a micro-computed tomographic method (micro-CT) and image processing after the fixation, decalcification, and dehydration of a mouse tongue. Results: 3D rendered images of tongue muscles obtained by micro-CT showed every muscle and their relationships to each other. The superior longitudinal and the hyoglossus muscles of the tongue made up one group, while the inferior longitudinal and the styloglossus muscles of the tongue made up another. The boundary of the two muscles in each group was difficult to distinguish. On the other hand, what appear to be newly described muscles were identified. These results indicate that our micro-CT method is beneficial and that classical knowledge of tongue muscles derived from two-dimensional (2D) images does not fully describe the actual complexity of the tongue muscles. In our opinion, 3D rendered images mixed with raw structure can provide a more in-depth picture of the tongue from an integrated as well as an analytical perspective.