Studies of the physiology and biomechanics of small ( approximately 1 cm) organisms are often limited by the inability to see inside the animal during a behavior or process of interest and by a lack of three-dimensional morphology at the submillimeter scale. These constraints can be overcome by an imaging probe that has sensitivity to soft tissue, the ability to penetrate opaque surfaces, and high spatial and temporal resolution. Synchrotron X-ray imaging has been successfully used to visualize millimeter-centimeter-sized organisms with micrometer-range spatial resolutions in fixed and living specimens. Synchrotron imaging of small organisms has been the key to recent novel insights into structure and function, particularly in the area of respiratory physiology and function of insects. X-ray imaging has been effectively used to examine the morphology of tracheal systems, the mechanisms of tracheal and air sac compression in insects, and the function of both chewing and sucking mouthparts in insects. Synchrotron X-ray imaging provides an exciting new window into the internal workings of small animals, with future promise to contribute to a range of physiological and biomechanical questions in comparative biology.