Increased atmospheric nitrogen (N) deposition is known to reduce plant diversity in natural and semi-natural ecosystems, yet our understanding of these impacts comes almost entirely from studies in northern Europe and North America. Currently, we lack an understanding of the threat of N deposition to biodiversity at the global scale. In particular, rates of N deposition within the newly defined 34 world biodiversity hotspots, to which 50% of the world's floristic diversity is restricted, has not been quantified previously. Using output from global chemistry transport models, here we provide the first estimates of recent (mid-1990s) and future (2050) rates and distributions of N deposition within biodiversity hotspots. Our analysis shows that the average deposition rate across these areas was 50% greater than the global terrestrial average in the mid-1990s and could more than double by 2050, with 33 of 34 hotspots receiving greater N deposition in 2050 compared with 1990. By this time, 17 hotspots could have between 10% and 100% of their area receiving greater than 15 kg N ha−1 yr−1, a rate exceeding critical loads set for many sensitive European ecosystems. Average deposition in four hotspots is predicted to be greater than 20 kg N ha−1 yr−1. This elevated N deposition within areas of high plant diversity and endemism may exacerbate significantly the global threat of N deposition to world floristic diversity. Overall, we highlight the need for a greater global approach to assessing the impacts of N deposition.