Water movement through the soil-plant-atmosphere continuum (SPAC) has long been regarded as a fundamental concept in studies of plant-water relations. Recent research suggests that water flux along the SPAC is more spatially and temporally variable than previously thought, occurring at night (nocturnal transpiration) as well as during the day and in multiple directions between plant roots and their surrounding soil (hydraulic redistribution). Interestingly, nocturnal transpiration and hydraulic redistribution are not well studied, particularly in grassland ecosystems. Here, we show that nocturnal water fluxes commonly occur in mesic tallgrass prairie. Using leaf gas exchange, stem sap flow, and stable isotope techniques, we characterized nighttime water movement between plants and the atmosphere and between plant roots and the soil at the Konza Prairie in northeastern Kansas, USA. At the leaf level, nocturnal transpiration rates vary among species and across the growing season (5-15% of total daily water flux). Nighttime transpiration rates are typically greatest in grasses and account for a larger proportion of total daily water-use than in forbs and shrubs at both the leaf and stem level. Conversely, shrubs show a greater tendency to redistribute water throughout the soil, although this phenomenon also varies across a growing season and is reduced by bison grazing. These results suggest that nocturnal water fluxes are seasonally variable, are influenced by management techniques such as grazing, and can account for a considerable portion of the total grassland water budget. Ultimately, these results highlight the importance of understanding these phenomena in greater mechanistic detail, as well as emphasize the need for better accounting of nocturnal fluxes in water budgets and ecosystem models.