Urban areas are among the land use types with the highes richness in plant species. A main feature of urban floras is the high proportion of non-native species with often divergent distribution patterns along urban–rural gradients. Urban impacts on plant species richness are usually associated with increasing human activity along rural-to-urban gradients. As an important stimulus of urban plant diversity, human-mediated seed dispersal may drive the process of increasing the similarity between urban and rural floras by moving species across urban–rural gradients. We used long motorway tunnels as sampling sites for propagules that are released by vehicles to test for the impact of traffic on seed dispersal along an urban–rural gradient. Opposite lanes of the tunnels are separated by solid walls, allowing us to differentiate seed deposition associated with traffic into vs. out of the city. Both the magnitude of seed deposition and the species richness in seed samples from two motorway tunnels were higher in lanes leading out of the city, indicating an ‘export’ of urban biodiversity by traffic. As proportions of seeds of non-native species were also higher in the outbound lanes, traffic may foster invasion processes starting from cities to the surrounding landscapes. Indicator species analysis revealed that only a few species were confined to samples from lanes leading into the city, while mostly species of urban habitats were significantly associated with samples from the outbound lanes. The findings demonstrate that dispersal by traffic reflects different seed sources that are associated with different traffic directions, and traffic may thus exchange propagules along the urban–rural gradient.