The field of ecohydrology is providing new theoretical frameworks and methodological approaches for understanding the complex interactions and feedbacks between vegetation and hydrologic flows at multiple scales. Here we review some of the major scientific and technological advances in ecohydrology as related to understanding the mechanisms by which plant–water relations influence water fluxes at ecosystem, watershed and landscape scales. We identify several cross-cutting themes related to the role of plant–water relations in the ecohydrological literature, including the contrasting dynamics of water-limited and water-abundant ecosystems, transferring information about water fluxes across scales, understanding spatiotemporal heterogeneity and complexity, ecohydrological triggers associated with threshold behavior and shifts between alternative stable states and the need for long-term data sets at multiple scales. We then show how these themes are embedded within three key research areas where improved understanding of the linkages between plant–water relations and the hydrologic cycle have led to important advances in the field of ecohydrology: upscaling water fluxes from the leaf to the watershed and landscape, effects of plant–soil interactions on soil moisture dynamics and controls exerted by plant water use patterns and mechanisms on streamflow regime. In particular, we highlight several pressing environmental challenges facing society today where ecohydrology can contribute to the scientific knowledge for developing sound management and policy solutions. We conclude by identifying key challenges and opportunities for advancing contributions of plant–water relations research to ecohydrology in the future.