To our knowledge, this was the first study to investigate the occurrence and distribution of antibiotics in the Karst river system in Kaiyang, Southwest China. Ten water samples were collected from the Karst river in Kaiyang, Southwest China. Thirty-five antibiotics, including nine sulfonamides, four tetracyclines, five macrolides, sixteen quinolones and chloramphenicol, were analyzed. The results suggest that antibiotics are widely prevalent in the Karst river, with macrolides and quinolones being the most dominant and occupying 47% and 43% of total antibiotic concentration, respectively. The maximum total concentrations of sulfonamides, tetracyclines, macrolides, and quinolones were 30.4, 421, 884, and 1,807 ng/L, respectively. Lincomycin, roxithromycin, nalidixic acid, ofloxacin, and norfloxacin were detected in all samples with a detection frequency of 100%. The main sources of antibiotics were wastewater treatment plants (WWTPs) and rural dumps that did not contain sanitary treatment, which accounted for 33% and 40% of the total antibiotics present in the Karst river. Due to an increase in river flow quantity, the presence of WWTPs and rural dumps did not affect the concentration and distribution of antibiotics in the Karst river; however, the mass flux of antibiotics were significantly affected by the contamination source and the poor natural attenuation.