Abstract: Karst underground rivers, which are widely distributed in karst areas in Southwest China, are important water resources. In recent years, with the continuous economic development, karst underground rivers have been rendered polluted to variable proportions. Nitrate pollution is a prominent and common problem in karst underground rivers. The high heterogeneity of karst aquifers makes it difficult to decipher the formation and distribution process of nitrate by simulation. The typical karst underground river system in Guancun Village, Daliang Town, Liuzhou City, Guangxi was selected as the study area. The land use was dominated by agriculture and the primary pollutant was nitrate. Multiple water samples were collected during the wet and dry seasons. The nitrate concentration and stable isotope variation characteristics were analyzed employing tracing and statistical methods. The results indicated that in the Guancun karst underground river system the trending of nitrate concentration during the wet and dry periods were consistent. The nitrate levels gradually increased from the Shanghu Cave to Xiahu Cave. On the contrary, the nitrate levels decreased from the Xiahu Cave to the underground river outlet. The waters of study area were obviously affected by human activities. The nitrates in the water primarily originated from agricultural and living sources. The input from the agricultural source had a profound influence on the nitrate concentration in the water. Due to the different sources of nitrate in the recharge water in different seasons, the distribution of nitrate concentration and stable water isotopes showed significant seasonal characteristics of the underground river system with the average nitrate concentration during wet season being greater than that during the dry season. As a result of the input of exogenous nitrate, mixing waters, and reduction processes, the distribution of nitrate concentration in karst underground river system also exhibit obvious spatial distribution characteristics. In this study, tracing method was employed to analyze the spatial and temporal distribution characteristics and the factors influencing nitrate levels in the karst underground river system. The conclusions arrived at would be significant in formulating non-point source pollution prevention strategies in karst areas.