Progress in high-resolution x-ray microtomography has provided us with a practical approach to determining three-dimensional (3D) structures of opaque samples at micrometer to submicrometer resolution. In this review, we give an introduction to hard x-ray microtomography and its application to the visualization of 3D structures of biological soft tissues. Practical aspects of sample preparation, handling, data collection, 3D reconstruction, and structure analysis are described. Furthermore, different sample contrasting methods are approached in detail. Examples of microtomographic studies are overviewed to present an outline of biological applications of x-ray microtomography. We also provide perspectives of biological microtomography as the convergence of sciences in x-ray optics, biology, and structural analysis.