Maintenance rails are an important passage for the safe operation and maintenance of bridges, significantly affecting the vortex-induced vibration (VIV) performance of main girders. A twin steel box girder cable-stayed bridge with the main span of 658 m was taken as the engineering background to investigate the influence mechanism of maintenance rails on the VIV performance of twin steel box girders. Combined with section model wind tunnel tests and two-dimensional fluid-structure interaction CFD numerical simulations, the vertical VIV performance of the main girder sections with and without maintenance rails was studied, and the mechanism of maintenance rails’ influence on VIV performance was analyzed. The results show that large vertical VIV occurs in main girder sections with maintenance rails, while the vertical VIV amplitude of the sections without maintenance rails decreases by more than 40%, indicating that maintenance rails significantly influence the VIV performance of the twin steel box girders. Periodic vortex shedding occurs on the upper and lower surfaces of main girder sections with maintenance rails, which results in a large pressure difference between the upper and lower surfaces of main girders, thus generating periodic vortex-induced force and causing large-amplitude vertical VIV of main girders. After removing the maintenance rails, the periodic vortex shedding pattern on the upper and lower surfaces of main girder sections with maintenance rails is disrupted, thereby suppressing the VIV amplitude of main girders.