A high pressure technique, called high-pressure cryocooling, has been developed as a method for biophysical and X-ray science studies. The method was developed primarily for crystal cryoprotection and dozens of macromolecular crystals have been successfully cryopreserved, including membrane protein crystals (e.g. a potassium ion channel). In addition, the method was successfully applied to stabilize ligand–protein interactions and to study the pressure effect on the structure of a yellow fluorescent protein, citrine. The procedure was then modified to entrap intermediate enzymatic states of human carbonic anhydrase. In this presentation, I will discuss the mechanism of high pressure cryocooling and its biophysical applications.