Interactive effects of reductions in plant species diversity and increases in atmospheric CO2 were investigated in a long-term study in nutrient-poor calcareous grassland. Throughout the experiment, soil nitrate was persistently increased at low plant species diversity, and CO2 enrichment reduced soil [NO3–] at all levels of plant species diversity. In our study, soil [NO3–] was unrelated to root length density, microbial biomass N, community legume contents, and experimental plant communities differed only little in total N pools. However, potential nitrification revealed exactly the same treatment effects as soil [NO3–], providing circumstantial evidence that nitrification rates drove the observed changes in [NO3–]. One possible explanation for plant diversity effects on nitrification lies in spatial and temporal interspecific differences in plant N uptake, which would more often allow accumulation of NH4+ in part of the soil profile at low diversity than in more species-rich plant communities. Consequently, nitrification rates and soil [NO3–] would increase. Elevated CO2 increased soil water contents, which may have improved NO3– diffusion to the root surface thereby reducing soil [NO3–]. Higher soil moisture at elevated CO2 might also reduce nitrification rates due to less aerobic conditions. The accordance of the diversity effect on soil [NO3–] with previous experiments suggests that increased soil [NO3–] at low species diversity is a fairly general phenomenon, although the mechanisms causing high [NO3–] may vary. In contrast, experimental evidence for effects of CO2 enrichment on soil [NO3–] is ambiguous, and the antagonistic interaction of plant species reductions and elevated CO2 we have observed is thus probably less universal.