The effects of temperature on N mineralization were studied in two organic surface horizons (LF and H) of soil from a boreal forest. The soil was incubated at 5 °C and 15 °C after adding 15 N and gross N fluxes were calculated using a numerical simulation model. The model was calibrated on microbial C and N, basal respiration, and KCl-extractable NH4+, NO3−, 15NH4+ and 15 NO3−. In the LF layer, increased temperature resulted in a faster turnover of all N pools. In both layers net N mineralization did not increase at elevated temperature because both gross NH4+ mineralization and NH4+ immobilization increased. In the H layer, however, both gross NH4+ mineralization and NH4+ immobilization were lower at 15 °C than at 5 °C and the model predicted a decrease in microbial turnover rate at higher temperature although measured microbial activity was higher. The decrease in gross N fluxes in spite of increased microbial activity in the H layer at elevated temperature may have been caused by uptake of organic N. The model predicted a decrease in pool size of labile organic matter and microbial biomass at elevated temperature whereas the amount of refractory organic matter increased. Temperature averaged microbial C/N ratio was 14.7 in the LF layer suggesting a fungi-dominated decomposer community whereas it was 7.3 in the H layer, probably due to predominance of bacteria. Respiration and microbial C were difficult to fit using the model if the microbial C/N ratio was kept constant with time. A separate 15N-enrichment study with the addition of glucose showed that glucose was metabolized faster in the LF than in the H layer. In both layers, decomposition of organic matter appeared to be limited by C availability.