Transport of nitrogen (N) in karst areas is more complex than in non-karst areas due to marked heterogeneity of hydrodynamic behaviour in the karst critical zone. Here, we present a novel, distributed, coupled hydrological-biogeochemical model that can simulate water and nitrogen transport in the critical zone of karst catchments. This new model was calibrated using integrated hydrometric, water stable isotope, and nitrogen-N concentration data at the outflow of Houzhai catchment in Guizhou province of Southwest China. Hydrological dynamics appears to control N load from the study catchment. Combining flow discharge and water stable isotopes significantly constrained model parameterisation and mitigate the equifinality effects of parameters on the simulated results. Karst geomorphology and land use have functional effects on spatiotemporal variations of hydrological processes and nitrogen transport. In the study catchment, agricultural fertilizer was the largest input source of N, accounting for 86% of the total. Plant uptake consumed about 45% of inputs, primarily in the low-lying valley bottom areas and the plain covered by relatively thick soils. Thus, a large amount of N released from soil reservoirs to the epikarst (via fractures or sinkholes) is then exported to the underground channel in the limestone area to the south. This N draining into groundwater could lead to extensive, potentially long-term contamination of the karst system. Therefore, improving the efficiency of fertilization and agricultural management in valleys/depressions is an urgent need to reduce N losses and contamination risk.