Intensive fertilization of grasslands with cattle slurry can cause high environmental nitrogen (N) losses in form of ammonia (NH3), nitrous oxide (N2O), and nitrate (NO3−) leaching. Still, knowledge on short-term fertilizer N partitioning between plants and dinitrogen (N2) emissions is lacking. Therefore, we applied highly 15N-enriched cattle slurry (97 kg N ha−1) to pre-alpine grassland field mesocosms. We traced the slurry 15N in the plant-soil system and to denitrification losses (N2, N2O) over 29 days in high temporal resolution. Gaseous ammonia (NH3), N2 as well N2O losses at about 20 kg N ha−1 were observed only within the first 3 days after fertilization and were dominated by NH3. Nitrous oxide emissions (0.1 kg N ha−1) were negligible, while N2 emissions accounted for 3 kg of fertilizer N ha−1. The relatively low denitrification losses can be explained by the rapid plant uptake of fertilizer N, particularly from 0–4 cm depth, with plant N uptake exceeding denitrification N losses by an order of magnitude already after 3 days. After 17 days, total aboveground plant N uptake reached 100 kg N ha−1, with 33% of N derived from the applied N fertilizer. Half of the fertilizer N was found in above and belowground biomass, while at about 25% was recovered in the soil and 25% was lost, mainly in form of gaseous emissions, with minor N leaching. Overall, this study shows that plant N uptake plays a dominant role in controlling denitrification losses at high N application rates in pre-alpine grassland soils.