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Anthropogenic nitrogen (N) input is known to alter plant and microbial α-diversity, but how N enrichment influences β-diversity of plant and microbial communities remains poorly understood. Using a long-term multilevel N addition experiment in a temperate steppe, we show that plant, soil bacterial and fungal communities exhibited different responses in their β-diversity to N input. Plant β-diversity decreased linearly as N addition increased, as a result of increased directional environmental filtering, where soil environmental properties largely explained variation in plant β-diversity. Soil bacterial β-diversity first increased then decreased with increasing N input, which was best explained by corresponding changes in soil environmental heterogeneity. Soil fungal β-diversity, however, remained largely unchanged across the N gradient, with plant β-diversity, soil environmental properties, and heterogeneity together explaining an insignificant fraction of variation in fungal β-diversity, reflecting the importance of stochastic community assembly. Our study demonstrates the divergent effect of N enrichment on the assembly of plant, soil bacterial and fungal communities, emphasizing the need to examine closely associated fundamental components (i.e., plants and microorganisms) of ecosystems to gain a more complete understanding of ecological consequences of anthropogenic N enrichment.