Nitrogen (N) fertilization effects on Eucalyptus growth and soil carbon (C) stocks are still controversial. We set up an N fertilization experiment in southern Brazil to evaluate initial tree growth and changes in soil organic matter (SOM). Four N levels (24-Reference, 36, 48 and 108 kg ha −1 of N) were tested and tree growth was assessed during the first two years. Afterwards, representative trees were chosen to evaluate fine-root biomass (FRB) and its spatial distribution. Soil was sampled to a 40-cm depth and SOM was fractionated in Particulate (POM) and Mineral-Associated Organic Matter (MAOM) for C and N content, and δ 13 C determination. Positive N effect on tree growth was seen only for tree height. N addition resulted in higher FRB. Changes in SOM were more expressive in top-soil layers. Overall, afforestation had positive effects on soil C. Increasing reference N dose resulted in higher C and N content in both SOM fractions. C and N dynamics were tightly correlated, especially in MAOM. Eucalypt-derived C was on average three-fold higher in POM. In summary, we showed that N fertilization may have positive but limited effects on tree growth, nevertheless it enhances fine-root biomass and C and N accumulation in SOM pools.