Agricultural crops with a C4 photosynthetic pathway rapidly expanded across North America as early as 800 A.D. Their distribution continues to expand globally as demands for food and biofuel production increase. These systems are highly productive, having a significant impact on carbon and water exchange between the land and atmosphere. Here, we investigate the relative impact of agricultural C4 vegetation on the 13CO2 photosynthetic discrimination and atmospheric isotopic forcing in the upper Midwest, United States. We address three questions: (1) What is the relative importance of C3 and C4 species to the CO2 budget? (2) How do these different photosynthetic pathways influence the photosynthetic discrimination within this heterogeneous landscape? (3) To what extent does land use change (i.e., a change in C4 crops) impact atmospheric isotopic forcing and the isotopic signature of the atmosphere? These questions are addressed using measurements obtained from the University of Minnesota tall tower (244 m) trace gas observatory (TGO) over the growing seasons of 2007 and 2008 and are supported with scaled-up values of discrimination and isotopic forcing based on ecosystem-scale eddy flux observations and high-resolution land use data. Our land use analyses indicate that local and regional C4 production was higher by 10% in 2007 due to increased demand for biofuel. The 2007 growing season was also characterized by moderate drought as a consequence of low antecedent soil water content. Isotopic flux ratio measurements from TGO provide evidence that the increase in C4 land use and drier soil conditions of 2007 had a significant impact on the growing season 13CO2 photosynthetic discrimination, which ranged from 11.5 to 14.8 in 2007 and 12.4‰ to 17.4‰ in 2008. Isotopic partitioning indicated that C4 species accounted for about 20 to 40% of the growing season gross photosynthetic CO2 exchange. The isoforcing analysis revealed that C3 discrimination dominated the atmospheric δa13 budget, especially during spring and fall. Estimates of 13CO2 photosynthetic discrimination for this region support recently published isotope modeling studies that explicitly accounted for increases in C4 cropland, which has significant implications for estimating the terrestrial carbon sink strength based on inverse modeling techniques.