Gross nitrogen (N) transformations are crucial in determining soil N status, but how gross N transformations change during post-agricultural succession remains poorly understood. Here, gross N transformations were measured using a 15N isotope dilution technique in a subtropical karst region, southwest China. A stratified random sampling scheme was adopted and the succession sequence included grassland (~4 years), shrubland (~15 years) and secondary forest (~30 years) with cropland as reference. The soil was leptosols (limestone soil). Soil total N concentrations were significantly (P < 0.05 hereafter) elevated in the shrubland and forest relative to the cropland and grassland. No clear pattern was found for NH4+ concentration, but NO3− concentration increased from the grassland to the forest. The gross rates of N mineralization and nitrification (GN) were significantly lower in the grassland than in the cropland, but increased significantly during post-agricultural succession. There were no clear patterns for the rates of dissimilatory NO3− reduction to NH4+ and gross NO3− immobilization. Gross NH4+ immobilization (GAI) in the forest was lowest but there was no significant difference among the cropland, grassland and shrubland. Gross N mineralization rate correlated significantly with protease activity, implying that the depolymerization of N-containing polymers was likely the rate-limiting step of gross N mineralization. Net nitrate production rate and GN:GAI ratio increased significantly from the grassland to the forest, supporting that soil N cycling likely became more open during post-agricultural succession in the karst region of southwest China.