Mercury mines in Guizhou province are the main base of mercury production and the most important resource base in China. The San-Dan mercury belt in Guizhou province contains a series of important mercury deposits. However, the source of metallogenic materials and the properties of metallogenic fluid of these mercury deposits have long been a controversial issue. In this study, we used cathode luminescence techniques to distinguish different stages of dolomite and calcite, laser ablation inductively coupled plasma mass spectrometry to analyze the trace elements, and stable isotope mass spectrometry techniques to analyze C–O isotopes compositions of dolomite and calcite in the Sixiangchang mercury deposit in San-Dan mercury belt. We also measured the sulfur isotope composition of cinnabar. Our study showed that dolomite can be divided into two stages, the lumpy dolomite associated with cinnabar in Dol 1 stage and dolomite vein in Dol 2 stage, which is associated with Cal 2 stage calcite vein. With the progress of mineralization, Al, As, Mo, Sb, and Sr elements were gradually enriched in the ore-forming fluid. The rare earth element (REE) partition curve of Dol 1 stage dolomite showed a trend of light REE enrichment. Cal 2 stage calcite and Dol 2 stage dolomite exhibited a flat-type REE partition curve, and Dol 2 stage dolomite showed a strong negative anomaly for Eu. δ¹³C of carbonate mineral variation ranges from ? 6.89 to ? 2.16 ‰, while δ¹⁸O variation ranges from 13.80 to 23.09 ‰, and the δ³⁴S variation range of cinnabar is 16.51–24.28 ‰. Carbonate mineral trace elements and C–O isotopes compositions suggested that early ore-forming fluid was reduced, and late ore-forming fluid was oxidized. The ore-forming fluid of the Sixiangchang mercury deposit is a mixture of deep crustal fluid and meteoric water in deep thermal circulation, and involved in the oxidation of organic matter. The cinnabar δ³⁴S results showed that sulfur mainly came from seawater sulfate with the participation of microbial reduction. Sulfur is sedimentary in origin and was derived mainly from the host-rock strata.