Globally, the aggregate length of roads exceeds 64 million km and 90.7% of protected areas are disturbed by road penetration, and therefore understanding how road networks impact species and ecosystem processes is highly relevant to applied conservation ecology. Among various effects on wildlife, roads may disrupt the interaction between plants and their animal-mediated seed dispersers, compromising forest regeneration and composition. Here, using the acorn-rodent system, we quantified to what extent rodent functions on seed dispersal are modified by roads over two years (i.e. 2013 and 2014). Road had negative effects on seed-dispersal services provided by rodents, with an approximate 30% reduction of seed dispersal effectiveness (SDE) and 50% reductions of dispersal distance in proximity to the road. These detrimental effects gradually declined along a gradient from the roadside towards forest interior. Declines of rodent abundance in proximity to the road seem to induce these depressed seed dispersal functions. Although the influential magnitudes of roads on SDE were similar in both years, its influential magnitudes on seed dispersal were stronger in 2014 which indicates the more negative impacts of road encroachment on seed dispersal services. Crucially, no tagged seeds were dispersed across the road, implying that it imposed a barrier effect on animal-mediated seed dispersal and plant recruitment. Given wildlife’s key roles in plant recruitment, we conclude that the ever-expanding effect of roads on animal-mediated seed dispersal may ultimately cause profound changes in forest composition and structure across diverse ecosystems, on a global scale.