Karst regions have long been recognised as landscapes of ecological vulnerability, however the mass balance and fate of mercury (Hg) in karst regions have not been well documented. This study focused on the largest contiguous karst area in China and investigated Hg mass balance in two catchments, one with high geological Hg (Huilong) and the other representative of regional background Hg (Chenqi). The mass balance of Hg was calculated separately for the two catchments by considering Hg in throughfall, open field precipitation, total suspended particulate matter (TSP), litterfall, fertilizer, crop harvesting, air-surface Hg⁰ exchange, surface runoff and underground runoff. Results show that litterfall Hg deposition is the largest loading (from atmosphere) of Hg in both catchments, accounting for 61.5% and 38.5% of the total Hg input at Huilong and Chenqi, respectively. Air-surface Hg⁰ exchange is the largest efflux, accounting for 71.7% and 44.6% of the total Hg output from Huilong and Chenqi, respectively. Because both catchments are subject to farm and forest land use, cultivation plays an important role in shaping Hg fate. Mercury loading through fertilizer was ranked as the second largest input (28.5%) in Chenqi catchment and Hg efflux through crop harvest was ranked as the second largest output pathway in both Huilong (27.0%) and Chenqi (52.9%). The net Hg fluxes from the catchments are estimated to be 1498 ± 1504 μg m⁻² yr⁻¹ and 4.8 ± 98.2 μg m⁻² yr⁻¹. The significantly greater magnitude of net Hg source in Huilong is attributed to higher air-surface Hg⁰ exchange. The output/input ratio of Hg in this study was much greater than has been reported for other forest or agricultural ecosystems and indicates that the karst region of Southwest China is a significant source of atmospheric Hg. The results of this study should be considered in the development of pollution control policies which seek to conserve fragile karst ecosystems characterised by high geological background of Hg.