The Brazilian savanna (Cerrado) has been extensively converted to croplands, pastures and forestry plantations, and the deforestation frontier continues expanding. Land conversion may cause critical changes in soil functioning, yet very little is still known about the impact of Cerrado conversion on nutrient cycling and soil fertility. Here, we addressed this knowledge gap by investigating the effects of the woodland cerrado (cerradão) conversion into pastures and Eucalyptus plantations on nitrogen availability and mineralization potential, considering a wide range of spatial and temporal variability due to soil depth, site conditions, and seasonal variation. For three sites in São Paulo state and each of the target land cover types, we assessed the total N and inorganic N (NH4-N and NO3-N) pools, potentially mineralizable nitrogen (PMN) and soil urease activity in the first 2 m of the soil profile. Cerrado conversion to either pastures or Eucalyptus plantations significantly reduced NH4-N, while NO3-N showed similar values in Cerrado and Eucalyptus and lower values in pastures. We found a consistent pattern of lower N mineralization in the uppermost soil layers associated to Cerrado conversion, with decreases in PMN rate and urease activity. The soil below 30 cm depth showed no relevant changes. Considering the first 30 cm of the soil profile, the reduction in the stocks of inorganic N (NH4-N + NO3-N) ranged from ~14% for the conversion to Eucalyptus to ∽20% for the conversion to pasture. The impact of land conversion on N cycling surpassed the influence of the spatial (between-site) and seasonal variation. Overall, the results indicate a decline in available N and overall soil fertility due to Cerrado conversion, which could further increase N limitation in the Cerrado region, increase fertilization needs for future exploitation, and compromise the recovery of Cerrado in case of land abandonment or restoration.