Partitioning of soil nitrogen (N) by niche separation among species may be an important mechanism explaining species coexistence and positive biodiversity-productivity relationships in terrestrial plant communities. However, there is little experimental evidence for such partitioning, in particular, as assessed across a gradient of species richness. In experimental communities of one, three, and six temperate grassland species in the field, we tested whether increasing species richness (1) decreases niche breadths of individual species, (2) decreases niche overlap among species, and (3) increases niche breadth of whole communities. Six N sources consisting of three different chemical forms of 15N-labeled N (15NO3(-), 15NH4+, 13C2-15N-glycine) injected at two soil depths (3 and 12 cm) were applied to each community. The chemical form and the soil depth of N characterize the niches for which niche breadth (Levins' B) and overlap (proportional similarity) were measured. After 48 hours, aboveground plant material was harvested to measure 15N enrichment. As expected, niche breadth of single species and niche overlap among species decreased with increased species richness, but community niche breadth did not increase. The decrease in niche breadth and niche overlap mostly occurred among subordinate species or pairs of subordinate and dominant species, rather than among dominant species. Species in the six-species mixtures mostly preferred NO3(-) from shallow soil. This may be partly explained by the presence of legumes in all six-species mixtures which allowed "N sparing" (i.e., increased availability of soil N since legumes rely more on atmospheric N2 than on soil N). Niche separation with respect to N uptake from different chemical forms and soil depths did not contribute much to facilitating the coexistence of dominant species, nor do our results suggest it as a major driver of positive diversity-ecosystem functioning relationships. However, partitioning of N may be important for the persistence of subordinate species.