The effects of soil-structure interaction on the seismic response of an isolated three span motorway overcrossing founded on piles are investigated by considering a real bridge located along the A14 Motorway in central Italy. The dynamic and mechanical properties of the soils are obtained from a comprehensive geotechnical characterization of the sites. Ten triplets of real accelerograms, defined at the outcropping bedrock, are adopted and processed by local response analyses to capture the site amplification effects and the free-field motions within the deposits. The soil-structure interaction effects are evaluated by means of the substructure method by comparing the seismic response of the structures with those obtained from conventional fixed base models. Analyses demonstrate that the soil-foundation dynamic compliance as well as the energy loss due to radiation damping dot not modify significantly the overall behaviour of the isolated bridges, while soil-structure interaction may increase deformations and forces on the isolation devices with respect to those obtained with fixed base models.