Winter biogeochemical processes have received considerable attention. Biological processes (e.g., microbial respiration and plant photosynthesis) do not cease, even at sub-zero temperatures. However, our knowledge of plant nitrogen (N) uptake at sub-zero soil temperatures is particularly limited for deciduous plant species, which do not have leaves during winter. We investigated plant N uptake by evergreen and deciduous species and soil N processes during sub-zero soil temperatures in cool temperate forest soil. Isotopically labelled nitrate (NO3-N-15) was injected into soil as a tracer of plant uptake and soil N dynamics at sub-zero temperature soil at a cool temperate field site. Over a period of 41 days, 6-48 mg/kg DW-1 of N-15 accumulated in evergreen species and deciduous tree species. Furthermore, the N-15 content in ammonium increased, suggesting ammonium production at sub-zero soil temperatures. The increase in (NH4)-N-15 was positively correlated with soil moisture, indicating an important role for soil water in N dynamics at sub-zero soil temperatures. Our findings demonstrate that N uptake by plants and soil N transformation did not cease at sub-zero soil temperatures. Further studies are needed to understand the importance of N dynamics at sub-zero soil temperatures.