Mine tailing pollution is an environmental problem that has attracted worldwide attention. Its large output and high pollution risks were serious threats to the environment. Sulfur oxidation processes under the mine tailing conditions can produce hydrogen ions, leading to the acidification of mine tailing, which will activate and migrate metal pollutants and further cause harmful effects to the surrounding environment. As important participants in the biogeochemical cycle of sulfur, sulfur oxidizing microorganisms are the main components of sulfur oxidation under the mine tailing condition. Therefore, it is particularly important to strengthen the understanding of in situ microbe-driven sulfur oxidation processes in mine tailings. Till now, however, isolation and identification of sulfur oxidizing microorganisms in mine tailing and their impacts on in-situ environment still need to be further explored. Thus, this study collected the mine tailing samples from the" world antimony capital" tin mine as the research object. The plate scribing method was used to isolate and purify the in-situ microbial population of mine tailing under the conditions of sulfur oxidation, and identify its species by using the 16S rRNA and sulfur oxidation gene (soxB) sequence analysis. Then, the microcosm experimental system of sulfur oxidation condition was constructed to verify the functions of two pure bacteria with sulfur oxidation genes. In addition, the relationship between mine tailing acidification and sulfur oxidizing microorganisms was explored by constructing the simulation experimental system of mine tailing in-situ conditions. The results showed that two pure strains involving sulfur oxidation genes were successfully obtained, which were named as Methyloversatilis sp. LJY-1 and Limnobacter sp. LJY-2. We found that LJY-1 was the first discovered Methyloversatilis species with sulfur oxidation function, and its sulfur oxidation ability was significantly stronger than that of LJY-2. Based on the simulation experiments of mine tailing in situ conditions, it was proved that the microbe-driven sulfur oxidation process significantly aggravated the acidification of mine tailing (P< 0.05). According to the differences in the water content of mine tailings, this study also revealed that the acidification process of mine tailing under flooding condition was significantly faster than that under the wet condition (P< 0.05). The study is of great significance to the management and restoration of mine tailing sites.