Atmospheric nitrogen (N) deposition has been identified as a major threat to biodiversity, but field surveys of its effects have rarely focussed on sites which are actively managed to maintain characteristic species. We analysed permanent quadrat data from 106 plots in nature reserves on calcareous grassland sites in the United Kingdom collected during a survey between 1990 and 1993 and compared the data with the results from resurvey of 48 of these plots between 2006 and 2009. N deposition showed no significant spatial association with species richness, species diversity, or the frequency of species adapted to low nutrient conditions in the 1990–1993 dataset. However, temporal analysis showed that N deposition was significantly associated with changes in Shannon diversity and evenness. In plots with high rates of N deposition, there was a decrease in species diversity and evenness, a decline in the frequency of characteristic calcareous grassland species, and a lower number of rare and scarce species. As all sites had active management to maintain a high diversity and characteristic species, our results imply that even focussed management on nature conservation objectives cannot prevent adverse effects of high rates of N deposition. Structural equation modelling was used to compare different causal mechanisms to explain the observed effects. For change in Shannon diversity, direct effects of N deposition were the dominant mechanism and there was an independent effect of change in grazing intensity. In contrast, for change in herb species number, indirect effects on soil acidity, linked to both N and S deposition, were more important than direct effects of N deposition.