Inorganic nitrogen (N) is the most important nutrient in soils, because it limits plant productivity and affects ecosystem function. It is produced by the mineralization of organic N to ammonium (NH4+) (MNorg) and the subsequent nitrification of NH4+ to nitrate (NO3−) (ONH4). Previous studies systematically evaluated the patterns and mechanisms of MNorg and ONH4 in soils on a global scale, but the characteristics of both processes in karst regions remain largely unknown. We conducted a15N-tracing study to investigate the rates of MNorg and ONH4, and the factors that influence these processes, from different natural ecosystems (mainly grass, shrub and forest) within karst regions. The rates of both MNorg and ONH4 exhibited large spatial variations, ranging from 0.42 to 23.8 mg N kg−1 d−1 and from 0.23 to 29.1 mg N kg−1 d−1, respectively, which were mainly ascribed to the heterogeneity of soil nutrients in karst regions. The concentrations of SOC, TN, soil inorganic N, NO3−, total K, Ca, Mg, WHC, and the rates of MNorg and ONH4 ranked as grass < shrub < forest, indicating that the improvement of soil conditions could stimulate the inorganic N production along the vegetation succession. The relative importance of the explanatory variables for inorganic N production suggested that the soil calcium concentration was the predominant determinant of the rates of MNorg and ONH4 in karst regions. A structural equation model indicated that soil calcium stimulates organic N accumulation, by serving as the substrate driving the increased rates of MNorg and ONH4 under high pH conditions. Overall, our results highlight the importance of soil calcium in determining the rates of MNorg and ONH4 in karst regions.