Background <p>Increasing atmospheric nitrogen (N) deposition is a major threat to plant diversity globally. Recent observations show that the reduced-to-oxidized (NH_x/NO_y) ratio of N deposition has been changing spatially and temporally. How and to what extent different N forms (i.e., NH_x and NO_y) influence grassland plant species loss are still unclear.</p> Methods <p>We employed a field manipulative experiment by using three N forms [i.e., Ca(NO₃)₂, NH₄NO_3, and (NH₄)₂SO₄] with six N addition levels (0, 4, 8, 16, 24, 32&#xa0;g&#xa0;N m⁻² year⁻¹) in a&#xa0;temperate grassland and conducted a greenhouse experiment culturing four plant species corresponding different plant functional groups under Ca(NO₃)₂ or (NH₄)₂SO₄ addition.</p> Results <p>Results from our field experiment showed that the plant species loss rate was greater under NH₄⁺–N than NO₃⁻–N enrichment. Plant species loss was driven by light asymmetry under NO₃⁻–N enrichment, while it was co-driven by light asymmetry and soil acidification under NH₄NO₃ enrichment. Under NH₄⁺–N enrichment, light asymmetry, pH decrease, NH₄⁺ toxicity, and metal toxicity jointly affected species loss. The greenhouse experiment provided direct evidence that legumes and forbs are more physiologically susceptible to NH₄⁺-induced toxicity than grasses.</p> Conclusions <p>Our results emphasize that N forms play a vital role in affecting grassland plant diversity. This suggests that regions with higher NH_x enrichment may experience more severe plant diversity losses as N deposition continues to increase. Therefore, appropriate measures should be adopted to mitigate species losses.</p>