The discovery that single molecules can be pulled has enabled unprecedented access to measurements of the energy landscape associated with structural transitions within proteins.However, some conformational changes, such as the closing of a substrate gate in an enzyme or the vertical collapse of a pore-forming protein during membrane insertion, cannot be probed in this way, since the applied force acts in a direction opposite to the structural movement.For these, what is needed is the controlled application of compressive forces to individual molecules.Here, by probing single complexes of the pore-forming cytotoxin, perfringolysin O, with compressive forces using atomic force microscopy, we catalyze a key structural transition of pore formation, the collapse of the D2 domain.Studying this transition with different force magnitudes and applied durations provides a means to measure basic properties of the energy landscape associated with this transition: the barrier height and position along the reaction coordinate.These values are in excellent agreement with predictions obtained from molecular dynamics simulations.We expect single molecule compression will become a generally effective tool to quantitatively study previously inaccessible conformational transitions in proteins.