The contamination of NO3− in groundwater is a global concern, requiring the identification of biogeochemical transformations and sources of nitrate (NO3−) to understand the different nitrogen (N) pathways. This is necessary for the regulation of dispersed pollution of groundwater caused by livestock and agricultural activities. Jinan, located in eastern China, serves as a suitable case study due to its reliance on karst groundwater as a water source and its significant population, industrial, and agricultural development, which pose a substantial risk of nitrogen pollutants in groundwater. Therefore, this study aims to investigate NO3− concentrations and isotopic compositions of N and oxygen in karst groundwater in Jinan City, Shandong province, eastern China, to determine the most significant sources and transformations of NO3−. Approximately 86% of the groundwater samples exceed the NO3−–N standard for drinking water proposed by the World Health Organization (WHO). Furthermore, chemical indicators (Cl−, HCO3−, and TDS) and the oxygen and nitrogen isotopic compositions of NO3− indicate that soil organic nitrogen, manure, and urban sewage are the main sources of NO3− in the groundwater. Isotopic analyses also suggest that various biogeochemical transformations, such as nitrification and volatilization, occur throughout the study area, with microbial nitrification being the dominant process in water resource areas with high NO3− concentrations. To reduce NO3− pollution in the Changqing–Xiaolipu water supply, several suggestions have been proposed.