Background and Aims: Fire and grazing are important disturbances in grasslands, yet we know little about how they impact a variety of plant physiological processes such as plant ecohydrology. Here, we assessed the impact of fire history and grazing by Bison bison on the source of water uptake and niche overlap in common grassland species at the Konza Prairie Biological Station, a temperate mesic grassland located in northeastern Kansas, USA. Methods: We used the stable isotopic signature of soil and xylem water to evaluate water uptake in Andropogo n gerardii, Vernonia baldwinii, Amorpha canescens,and Rhus glabra within varying grazing (grazed, ungrazed), fire (0,1,2 or 3 years since last burn), topography (upland, lowland), and month (July, August) contrasts over 3 years (2013–2015). Results: The presence of grazers, not fire history, altered water uptake patterns in these common grassland species. Particularly, grazing increased the proportion of shallow water utilized by A. gerardii and R. glabra, reducing niche overlap with other co-occurring species. However, these responses varied intra-annually and were often modulated by topography. Conclusions: These results suggest that grazing can alter aspects of grassland ecohydrology at small scales, which may extend to impact community and ecosystem processes at larger spatial scales.