Rates of biological nitrogen fixation (BNF) were measured in ecosystem. In this study, we measured rates of BNF in ecosystem compartments (bulk soil, forest floor, rhizosphere soil, and nodule) in two mature tropical plantations in southern China with legume trees ( Acacia auriculiformis, AA) and with non-legume trees ( Eucalyptus urophylla, EU) after 4 years of nitrogen (N) fertilization (0, 50, and 100 kg N ha year). BNF rates of bulk soil were comparable between plantations, while rates of rhizosphere soil were significantly higher in the EU plantation and rates of forest floor were significantly higher in the AA plantation. Thus, total BNF rates were comparable between plantations (AA = 6.04 kg N ha year; EU= 6.42 kg N ha year). In the AA plantation, N addition significantly decreased BNF rates in all measured compartments and thus the total rates. In the EU plantation, N addition did not change BNF rates of forest floor, but significantly decreased rates of bulk soil and increased rates of rhizosphere soil; thus, total rates did not change. Our findings provide evidence that forest type is an important factor regulating the effects of external N input on BNF, and suggest that elevated atmospheric N deposition in recent decades will suppress total N fixation in mature forests with legume trees but not in those with non-legume trees.