Background and aims: The amount of nitrogen (N) derived from symbiotic N₂ fixation by legumes in grasslands might be affected by anthropogenic N and phosphorus (P) inputs, but the underlying mechanisms are not known. Methods: We evaluated symbiotic N₂ fixation in 17 natural and semi-natural grasslands on four continents that are subjected to the same full-factorial N and P addition experiment, using the ¹⁵N natural abundance method. Results: N as well as combined N and P (NP) addition reduced aboveground legume biomass by 65% and 45%, respectively, compared to the control, whereas P addition had no significant impact. Addition of N and/or P had no significant effect on the symbiotic N₂ fixation per unit legume biomass. In consequence, the amount of N fixed annually per grassland area was less than half in the N addition treatments compared to control and P addition, irrespective of whether the dominant legumes were annuals or perennials. Conclusion: Our results reveal that N addition mainly impacts symbiotic N₂ fixation via reduced biomass of legumes rather than changes in N₂ fixation per unit legume biomass. The results show that soil N enrichment by anthropogenic activities significantly reduces N₂ fixation in grasslands, and these effects cannot be reversed by additional P amendment.