Iowa's tallgrass prairies, the once dominant vegetation, were replaced by crops so that currently, only 0.1% of native prairies remain and approximately 80% of the land is dedicated to corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) production. This dramatic land use change has caused severe hydrological alterations (i.e. surface runoff, sediment and nutrient export) affecting the productivity and water quality of the region. In the last years, incorporation of prairie vegetation within agriculturally dominated watersheds has been demonstrated to have positive effects on restoring hydrologic balance by increasing soil water infiltration and nutrient stabilization. However, less attention has been given to the impact that plant water use from native perennial vegetation might also have on stabilizing hydrologic processes. This study aimed to increase our knowledge about water use patterns in native prairies and crops, at both the stand (evapotranspiration) and plant (transpiration) level, in order to enhance our understanding of the potential role of introduced prairie ecosystems on hydrologic regulation. To accomplish the objective, two field studies were conducted at the Neal Smith National Wildlife Refuge located in Central Iowa, and one study was conducted in the Forestry greenhouse at Iowa State University. Micrometeorological, thermometric and physiological techniques were used to measure plant water use rates and understand water use patterns. Overall, prairie vegetation exhibited higher evapotranspiration than crop fields early and late in the season, suggesting a higher efficacy by the former to increase the soil water storage capacity during typical wet springs, which would help to reduce runoff. At the plant level, transpiration of native prairie species higher responsiveness than crops to changes in soil water content and a significant increment in water uptake was observed following water inputs preceded by dry periods at both field and greenhouse conditions. We conclude that prairie vegetation can help to restore hydrologic processes in agricultural landscapes by extracting more water from the soil when soil water holding capacity is most needed.