Diaphragm cutouts are set to release redundant constraints and hence reduce weld fatigue at the connection of U-ribs to diaphragms in orthotropic steel decks. However, most fatigue cracks which originate from the edge of cutouts are in fact detected in the diaphragms. Therefore, a retrofit technology on cracked cutouts at the diaphragm is proposed and applied to the orthotropic steel box girder of a suspension bridge. Firstly, the stress concentration on the cutout is analyzed through refined finite element analyses. Furthermore, the fatigue cracked cutouts are retrofitted by changing their geometrical parameters. Thereafter, an optimized geometry and the size of diaphragm cutouts were confirmed and applied in the rehabilitation of a suspension bridge. On-site wheel load tests were carried out before and after retrofitting of the diaphragm cutout. The stress distributions along the edges of the cutouts and at the side of a diaphragm were measured under a moving vehicle. The stress spectra at two critical locations on the edge of a cutout was obtained under longitudinally and laterally moving vehicles. Finally, the fatigue life of the cutouts is assessed by the modified nominal stress method. The analytical and test results indicate that the wheel loads on the deck transmit stress to the diaphragms through the U-ribs, during the load transmission process, the stress flow is obstructed by diaphragm cutouts, resulting in local stress concentrations around the cutouts. In addition, the overall size of the cutouts should be small, but the radius of the transition arc should be large, thus the stress flow will not be obviously obstructed. After the retrofitting of the cutouts by improved geometry, the maximum stress decreases by 87.6 MPa, which is about 40% of the original stress. The equivalent constant amplitude stress is reduced by 55.2% when the lateral position of the wheel loads is taken into consideration. Based on the stresses obtained by finite element analysis (FEA) and experimental tests, the fatigue lives of the original cutouts are 1.7 and 4.9 years, respectively, which increase to 78.1 and 155.5 years, respectively, after the cutouts were retrofitted, which indicates that the improved geometry and retrofit technology can enhance the fatigue performance and extend the fatigue life of diaphragm cutouts with fatigue cracks.