A system and method for immobilization of the human breast in a prone position during imaging and radiotherapy. The system includes a vacuum-assisted breast immobilization cup comprising two layers joined together with an air space between. The inner layer has densely perforated holes while the outer layer is airtight. When a negative vacuum is applied to the intermediate airspace the breast tissue is sucked against the mesh wall of the inner layer to preventing sliding. In addition, the cup assembly is locked into a fixed position against the patient torso via a body fixture, as well as against the patient support structure. The established geometric relationship among the cup, chest wall, imaging/treatment table or couch may be accurately repeated. The system further includes a three-dimensional localization system to uniquely place the breast in a coordinate system having fixed geometric relationship with the breast immobilization cup.