Van der Waals (vdW) interaction, and its subtle interplay with chemically specific interactions and surface roughness at metal/organic interfaces, is critical to the understanding of structure–function relations in diverse areas, including catalysis, molecular electronics and self-assembly1, 2, 3. However, vdW interactions remain challenging to characterize directly at the fundamental, single-molecule level both in experiments and in first principles calculations with accurate treatment of the non-local, London dispersion interactions. In particular, for metal/organic interfaces, efforts so far have largely focused on model systems consisting of adsorbed molecules on flat metallic surfaces with minimal specific chemical interaction4, 5, 6, 7, 8, 9. Here we show, through measurements of single-molecule mechanics, that pyridine derivatives10, 11 can bind to nanostructured Au electrodes through an additional binding mechanism beyond the chemically specific N–Au donor–acceptor bond. Using density functional theory simulations we show that vdW interactions between the pyridine ring and Au electrodes can play a key role in the junction mechanics. These measurements thus provide a quantitative characterization of vdW interactions at metal/organic interfaces at the single-molecule level.