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In the Central Great Plains of North America, fire suppression is causing transitions from grasslands to shrublands and woodlands. This woody encroachment alters plant community composition, decreases grassland biodiversity, undermines key ecosystem services, and is difficult to reverse. How native grazers affect woody encroachment is largely unknown, especially compared to domesticated grazers. Bison were once the most widespread megafauna in North America and are typically categorized as grazers, with negative effects on grasses that indirectly benefit woody plants. However, bison can negatively impact woody plants through occasional browsing and mechanical disturbance. This study reports on a 30-year experiment at Konza Prairie Biological Station, a mesic grassland in the Central Great Plains of North America, under fire suppression and experimental presence/absence of bison. Based on remote sensing, deciduous tree canopy cover was lower with bison (6% grazed vs. 16% ungrazed). Shrub land cover showed no difference (42% grazed vs. 41% ungrazed), while herbaceous land cover was higher with bison (51% grazed vs. 40% ungrazed). Evergreen tree canopy cover (Juniperus virginiana L.), which decreases biodiversity and increases wildfire risk, was approximately 0% with bison compared to 4% without bison. In the survival trial of J. virginiana seedlings, we found a 40% overwinter mortality with bison, compared to 5% mortality without bison. Compared to ungrazed areas, native plant species richness was 97% and 38% higher in bison-grazed uplands and lowlands, respectively. Species evenness and Shannon's index were higher in the bison treatment in uplands, but not in lowlands. Bison affected community composition, resulting in higher cover of short grass species and lower tree cover. While grazers are generally assumed to favor woody plants, we found that bison had the opposite effect at low fire frequencies. We argue that the large size of bison and their behaviors account for this pattern, including trampling, horning, and occasional browsing. From a conservation perspective, bison might hamper tree expansion and increase plant diversity in tallgrass prairies and similar grasslands.