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Abstract We introduce a new “ecosystem‐scale” experiment at the Cedar Creek Ecosystem Science Reserve in central Minnesota, USA to test long‐term ecosystem consequences of tree diversity and composition. The experiment—the largest of its kind in North America—was designed to provide guidance on forest restoration efforts that will advance carbon sequestration goals and contribute to biodiversity conservation and sustainability.The new Forest and Biodiversity (FAB2) experiment uses native tree species in varying levels of species richness, phylogenetic diversity and functional diversity planted in 100 m²and 400 m²plots at 1 m spacing, appropriate for testing long‐term ecosystem consequences. FAB2 was designed and established in conjunction with a prior experiment (FAB1) in which the same set of 12 species was planted in 16 m²plots at 0.5 m spacing. Both are adjacent to the BioDIV prairie‐grassland diversity experiment, enabling comparative investigations of diversity and ecosystem function relationships between experimental grasslands and forests at different planting densities and plot sizes.Within the first 6 years, mortality in 400 m²monoculture plots was higher than in 100 m²plots. The highest mortality occurred inTilia americanaandAcer negundomonocultures, but mortality for both species decreased with increasing plot diversity. These results demonstrate the importance of forest diversity in reducing mortality in some species and point to potential mechanisms, including light and drought stress, that cause tree mortality in vulnerable monocultures. The experiment highlights challenges to maintaining monoculture and low‐diversity treatments in tree mixture experiments of large extent.FAB2 provides a long‐term platform to test the mechanisms and processes that contribute to forest stability and ecosystem productivity in changing environments. Its ecosystem‐scale design, and accompanying R package, are designed to discern species and lineage effects and multiple dimensions of diversity to inform restoration of ecosystem functions and services from forests. It also provides a platform for improving remote sensing approaches, including Uncrewed Aerial Vehicles (UAVs) equipped with LiDAR, multispectral and hyperspectral sensors, to complement ground‐based monitoring. We aim for the experiment to contribute to international efforts to monitor and manage forests in the face of global change.