The effects of the establishment of strips of native prairie vegetation (SNPV) in agricultural fields on soil water dynamics and depth of plant water uptake by annual (ROWCROP) and perennial vegetation were studied. Soil water storage (SWS) was monitored biweekly to a depth of 100 cm during the growing seasons of 2007, 2008, 2009 and 2010 and differences were analyzed by land cover, topographic position and season primarily. Depth of water uptake (DWU) was assessed during the growing season of 2010 in four prairie species (Ratibida pinnata, Bromus ciliatus, Elymus canadensis, and Andropogon gerardii), and one crop species (Zea mays). DWU was analyzed by species, topographic position, photosynthetic pathway (C₃ or C₄), and by soil moisture available in the topsoil. DWU was assessed using stable isotopes of oxygen δ18O and hydrogen δD, and a calibration process was developed for vacuum cryogenic distillation to extract water from clay rich soils for isotopic analysis. Our results indicated that SWS was lower under prairie vegetation than ROWCROP after one year of establishment of the SNPV, with differences among land covers more pronounced in the first 30 cm of soil. During years of low and high precipitation the differences in SWS among land covers were minimal, which leads us to propose a threshold effect. Our data indicates that all species obtained water from the first 70 cm of soil. DWU was not influenced by the relative topographic position of individual plants but was influenced by the soil moisture available in the topsoil. Analyzed by photosynthetic pathway, C₃ plants showed more sensitivity to changes in soil moisture, shifting to deeper depths than C₄ when soil moisture was limiting in the topsoil.