Abstract: With the development of the binary structure of karst landforms, surface water is largely drained with rapid loss of nutrients. However, the pathway and mechanism of nutrient loss remain elusive. From a three-dimensional (vegetation-soil-epikarst system) perspective of a critical zone in karst area, this study conducted rainfall induced runoff and nitrogen loss monitoring during monsoon in karst shrub slopes. Isotope-based (D and ¹⁸O) hydrograph separation method was applied to partition the ratio of ‘old’ and ‘new’ water in main hydrological path. The main results were summarized as follows. Deep percolation and interflow were the dominant hydrological pathways, accoun-ting for 71% and 9% of total rainfall amount, respectively. In contrast, surface runoff occupied less than 2%. Both deep percolation and interflow were dominated with 85% and 61% of old water, respectively. The highest nitrate concentration occurred in deep percolation (1.97 mg·L^-1), while the highest ammonium nitrogen concentration occurred in interflow (1.18 mg·L^-1). Deep percolation contributed 89.4% of total nitrogen loss, which was significantly higher than that of surface runoff and interflow. Old water ratio showed a significant positive correlation with nitrate nitrogen concentration, ammonium nitrogen concentration, and total nitrogen loss, suggesting it might be the main agent driving nitrogen migration for the whole soil-epikarst system in karst hillslopes. The results would provide scientific basis for rational allocating water resources and developing nutrient loss control technology in karst region of southwestern China.