This work investigates the effects of soil-structure interaction and spatial variability of seismic motion due to nonlinear site amplification on the seismic behaviour of long multi-span bridges founded on piles. An analysis framework able to include the spatial variation of ground motion induced by specific geological and geomorphological scenarios in the seismic soil-structure interaction analysis of long bridges is adopted, exploiting advantages of the substructure approach. The methodology is applied to a case study constituted by a pile-supported multi-span bridge founded in a soft clay deposit overlaying a stiff bedrock with three different configurations: horizontal, inclined and wedge-shaped. The reference input motion at the outcro...