The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work.