The North American prairie covered about 3.6 million-km(2) of the continent prior to European contact. Only 1-2% of the original prairie remains, but the soils that developed under these prairies are some of the most productive and fertile in the world, containing over 35% of the soil carbon in the continental United States. Cultivation may alter microbial diversity and composition, influencing the metabolism of carbon, nitrogen, and other elements. Here, we explored the structure and functional potential of the soil microbiome in paired cultivated-corn (at the time of sampling) and never-cultivated native prairie soils across a three-states transect (Wisconsin, Iowa, and Kansas) using metagenomic and 16S rRNA gene sequencing and lipid analysis. At the Wisconsin site, we also sampled adjacent restored prairie and switchgrass plots. We found that agricultural practices drove differences in community composition and diversity across the transect. Microbial biomass in prairie samples was twice that of cultivated soils, but alpha diversity was higher with cultivation. Metagenome analyses revealed denitrification and starch degradation genes were abundant across all soils, as were core genes involved in response to osmotic stress, resource transport, and environmental sensing. Together, these data indicate that cultivation shifted the microbiome in consistent ways across different regions of the prairie, but also suggest that many functions are resilient to changes caused by land management practices – perhaps reflecting adaptations to conditions common to tallgrass prairie soils in the region (e.g., soil type, parent material, development under grasses, temperature and rainfall patterns, and annual freeze-thaw cycles). These findings are important for understanding the long-term consequences of land management practices to prairie soil microbial communities and their genetic potential to carry out key functions.