A large amount of cropland in the Southwestern (SW) China karst area has been abandoned since the implementation of the Grain for Green project at the end of the last century, which significantly impacted the biogeochemistry cycle in soil. However, the effect of cropland abandonment on the soil nitrogen (N) cycle and the N loss process is still little understood. N content and isotope analysis were used to assess karst soil’s N fate and explore the N loss process in SW China. Soil samples were collected from four land uses, cropland (CR), abandoned CR (AC), shrubland and grassland (SG), and secondary forest (SF), by seasonal in a typically mixed land-use karst catchment at six soil depths. The results showed that the average soil particulate organic N (PON) content increased from 1.58 ± 0.49 g kg−1 to 2.70 ± 1.82 g kg−1, whereas average dissolved nitrate (NO3–-N) content decreased from 10.77 ± 8.87 mg kg−1 to 1.72 ± 1.84 mg kg−1 during revegetation. There was a decreasing amount of PON and dissolved N (NH4+-N、NO3–-N、TDN) content from surface to the bottom of the soil profile and large variation mainly occurred in 0 – 30 cm in the soil profiles of four different land uses, which suggests N transformation mainly occurred in topsoil (<30 cm). Mineralization was the major nitrogen transformation process that affected PON content and δ15N-PON values. The highest δ15N-PON fractionation (-5.0‰) in the soil profiles of the four types of land use caused by mineralization was detected in SF. Variation of N content and isotope values in soil profiles after cropland abandonment suggested that the loss of NO3–-N in karst catchment mainly came from the topsoil of cropland. Overall, N loss from the topsoil of cropland should pay more attention, while cropland abandonment can effectively reduce the loss of dissolved N from the thin karst soil layer.