Roads promote different positive and negative effects on biodiversity, and consequently on species interactions (mutualism, predation, parasitism, competition, commensalism, and amensalism). Yet, such impact is seldom considered in Road Ecology. In this chapter, we synthesize the current knowledge on the effects of roads on species interactions and suggest future avenues of research to fill current knowledge gaps on the impacts of transport infrastructure on ecological networks. Most road-related effects on interactions are mediated by habitat modification, pollution (noise), changes in the chemical environment, barrier effect, and increased mortality due to roadkill. Nevertheless, we are far from understanding in what conditions and to what degree are the different interactions affected. Research should address the effects of roads on ecological networks, ecosystem functioning, and ecosystem services.