To address the practical problem of non-uniform wind speed distribution encountered by large-span cable-stayed bridges crossing complex terrains, the paper takes a large-span separated steel box girder cable-stayed bridge as the research project, and the bridge structural model is established using finite element method. Two forms of wind speed distribution along the bridge are considered: parabolic distribution and linear distribution. Time-domain analysis of the bridge buffeting responses is conducted under different distribution forms, and compared with uniform wind speed distribution, to determine the effect of non-uniform distribution on the buffeting responses. The results show that compared to uniform distribution of wind speed, non-uniform distribution of wind speed significantly increases the lateral displacement, vertical displacement, and torsion angle response of the bridge overall. Under parabolic distribution, the lateral displacement of the main span exhibits the parabolic shape, and the double peak phenomenon appears in the vertical displacement in the range of the main span. Under linear distribution, the displacement response on the right side of the main span significantly increases, and the lateral response of the main span does not show the symmetrical distribution, while the vertical displacement exhibits the double peak as well.