Karst aquifers, a significant source of drinking water worldwide, are vulnerable to anthropogenic pollutants because of their special hydrogeological conditions. However, distinguishing the nitrogen transformation process is difficult owing to the heterogeneity of karst media. Therefore, the process of nitrogen transformation in different karst aquifer media remains poorly understood. In this study, multiple stable isotopic and physicochemical indicators are combined, the sources of water and nitrogen are analysed, and the degree of openness and redox conditions of different media are discussed to elucidate the nitrate sources and N transformation in groundwater with different degrees of openness. Water isotopes indicate local meteoric groundwater origin. However, different karst aquifer media have different degrees of openness, resulting in the highest deuterium–oxygen isotope values in surface water, followed by conduit water, fissure water, and isolated cave water. Based on the evidence from isotope and physicochemical indicators, different karst aquifer media have different redox processes. Isolated cave water environments were under anoxic conditions dominated by sulfate reduction reactions. Fissure water was under temporary anoxic or sub-anoxic conditions dominated by denitrification reactions. Conduit water and karst surface water were between micro-oxygen and oxidising conditions, and nitrification was the key process. This phenomenon was observed because the diversity of karst media produces different conservation conditions and openness. The nitrate isotope and Bayesian mixing model indicate that the main sources of nitrate in karst groundwater in the study area are mostly associated with manure and sewage wastes, and a few are from soil organic nitrogen, atmospheric deposition, and chemical fertilizer. This study helps identify the migration and transformation patterns and processes of nitrogen in different karst aquifer media, providing a scientific basis for the restoration and remediation of contaminated karst groundwater.