Permafrost regions contain a substantial fraction of Earth’s soil nitrogen, which is vulnerable to climate change. The response of this crucial N stock to warming could impact the permafrost–climate feedback by altering plant productivity, microbial decomposition and nitrous oxide emissions. However, the long-term trajectory of soil N stocks in response to warming remains unclear. Here we present results from a ten-year field warming experiment in a permafrost ecosystem on the Tibetan Plateau. We made repeated measurements of soil N stocks to 50 cm depth and assessed 28 N-cycling variables to explore three primary pathways affecting soil N stocks, including N inputs, microbial N transformations and N losses. Our results reveal that, despite no changes being observed during the initial years of the experiment, warmed plots experienced a decline in surface soil N stocks (an average 7.7% reduction relative to control plots) after eight years of warming. This decrease is associated with the enhanced N sequestration in perennial plant biomass, increased ecosystem N leaching and gaseous N losses from soils. Our findings underscore the vulnerability of soil N stocks in permafrost regions to ongoing warming, and suggest that the potential permafrost–climate feedback may be stronger than previously anticipated.