To explore the influence of spatially varying ground motion on the dynamic behavior of a train passing through a three-tower cable-stayed bridge, a 3D train–track–bridge coupled model is established for accurately simulating the train–bridge interaction under earthquake excitation, which is made up of a vehicle model built by multi-body dynamics, a track–bridge finite element model, and a 3D rolling wheel–rail contact model. A conditional simulation method, which takes into consideration the wave passage effect, incoherence effect, and site-response effect, is adopted to simulate the spatially varying ground motion under different soil conditions. The multi-time-step method previously proposed by the authors is also adopted to improve computational efficiency. The dynamic responses of the train running on a three-tower cable-stayed bridge are calculated with differing earthquake excitations and train speeds. The results indicate that (1) the earthquake excitation significantly increases the responses of the train–bridge system, but at a design speed, all the running safety indices meet the code requirements;(2) the incoherence and site-response effects should also be considered in the seismic analysis for long-span bridges though there is no fixed patt for determining their influences;(3) different train speeds that vary the vibration characteristics of the train–bridge system affect the vibra-tion frequencies of the car body and bridge.