<p>The hydrological cycle in karst subterranean river basins is complex and concealed, posing challenges to conventional monitoring and sustainable water management. This study investigates the hydrogen and oxygen isotopic characteristics in a typical subterranean river basin within the karst region of Southwest China, to elucidate seasonal water sources and hydrological processes. Results reveal distinct seasonal isotopic patterns in karst water, with the degree of isotopic enrichment decreasing in the following order: Transitional seasons (characterized by moderate precipitation and stable hydrological conditions), the wet season and then the dry season. Natural isotopic fractionation is limited, with dry-season samples showing a slightly stronger influence of evaporation. Spatially, spring water δ¹⁸O exhibits an elevation effect, and subterranean river water shows progressive isotopic depletion downstream. The karst vadose zone significantly buffers and mixes precipitation signals, leading to more stable isotopic compositions in karst water. Bayesian mixing analysis indicates that baseflow (from epikarst springs, descending springs, and surface streams) is the dominant recharge source for the subterranean river, with direct precipitation contribution being limited (&lt;10%). The study delineates unique dual hydrological pathways in Southwest China’s karst: A rapid conduit-flow pathway for storm runoff and a gradual infiltration pathway that sustains baseflow. This mechanism enables effective seasonal regulation and efficient utilization of water resources. These findings enhance the understanding of karst hydrological cycles and demonstrate the utility of isotope techniques in deciphering complex flow processes within subterranean river basins.</p>