Soil nitrogen (N) availability is crucial in governing the dynamics of ecosystem structure and function. However, the variation pattern of soil N availability during post-agricultural succession has been poorly understood for karst regions. Here, soil N availability variables, including concentrations of soil total N (TN), dissolved inorganic N (DIN=ammonium (NH4+)+nitrate (NO3−)), dissolved organic N (DON), rates of net N mineralization and nitrification at different successional stages, i.e. cropland, grassland, shrubland and secondary forest were investigated at a regional scale, in order to get a general pattern of soil N availability dynamics following agricultural abandonment in the karst region of southwest China. Concentrations of soil TN, soil NO3−, DIN, net N mineralization and nitrification rates increased from grassland to forest. Soil NO3− concentration in secondary forest (37.71±5.01mgNkg−1) reached the level in cropland (31.01±2.93mgNkg−1). Soil NO3−:NH4+ ratio in grassland was not significantly different from 1, but increased directly and reached 13.02±1.16 in secondary forest, equivalent to the level in cropland (15.15±1.98). The dynamics of soil DIN:DON ratio followed the same pattern as soil NO3−:NH4+ ratio. Our study suggests that soil N availability increases and soil N cycling becomes progressively open following agricultural abandonment in the karst region, southwest China. •Soil N availability increases along post-agricultural succession in karst regions.•N cycle becomes more open as succession proceeds.•Ca and Mg are important contributors to the increase of soil N availability.