<p>Under the fragile ecological conditions characteristic of karst terrains, the synergistic effects of soil erosion and anthropogenic activities have significantly intensified sulfate (SO₄^2-) mobilization and dispersion, profoundly influencing aquatic ecosystem stability and regional carbon cycling dynamics. To systematically investigate SO₄^2- geochemical behavior mechanisms in such environments, this study focused on the Chiwu Creek Basin, a representative karst agricultural basin. By integrated water chemistry analysis with multi-isotope tracing techniques (δ³⁴S_SO4, δ¹⁸O_SO4, δD and δ18O_H2O) and Bayesian mixing modeling (Simmr) to characterize hydrochemical features and quantitatively apportion SO₄^2-sources and transformation processes. Key findings reveal: Distinct karst hydrochemical signatures (HCO₃-Ca·Mg type) with rainfall dilution driving pronounced seasonal SO₄^2-concentration variations; Dominant sulfate contributions from gypsum dissolution (surface water 49.7% &gt; groundwater 28.7%) and soil sulfates (groundwater 33.3% &gt; surface water 28.7%), exhibiting marked wet-season increases that underscore rainfall-erosion coupling in sulfur cycling; Substantial uncertainty (UI₉₀) in natural source contributions (31.3–34.4%) versus more stable anthropogenic inputs (&lt; 12% combined), highlighting natural process amplification in sulfate geochemistry. While anthropogenic sources (sewage/fertilizers/detergents) constitute minor proportions, their environmental significance remains non-negligible. These insights advance fundamental understanding of karst sulfur biogeochemistry while providing critical scientific support for water resource management and carbon sink quantification in Southwest China’s ecologically sensitive karst regions.</p>