The precise identification of the aerodynamic loads on vehicles is a fundamental concern in assessing the ride comfort of vehicles crossing long-span bridges in the presence of strong crosswinds. However, the consideration of aerodynamic interference between vehicles and truss girder bridges is limited in existing studies. Furthermore, in practical engineering, wind barriers are commonly installed to enhance the ride comfort of vehicles crossing long-span bridges, but previous studies have primarily focused on the impact of wind barriers on the aerodynamic coefficients and dynamic responses of vehicles. This paper focuses on the aerodynamic interaction between trucks and truss girder bridges, through wind tunnel testing. The aerodynamic coefficients of the vehicle with three different ventilation ratios and four different heights of wind barriers were obtained. The dynamic response of road vehicles on a truss girder suspension bridge with a main span of 965 m was numerically calculated using the coupled analysis of wind-vehicle-bridge system and measured aerodynamic coefficients considering aerodynamic interference. On this basis, the comfort of road vehicles was investigated with different section types of the girder and different wind barrier parameters, and it was evaluated accordance with the overall vibration total value (OVTV) recommended in the ISO 2631–1. It is found that the aerodynamic coefficients of road vehicles are sensitive to the type of wind barrier. Wind barriers can reduce the road vehicle’s aerodynamic loads at high wind yaw angles. Wind barriers can also improve the ride comfort of road vehicles on the long-span truss girder suspension bridge.