Uncovering the factors influencing antimony (Sb) species in sediments is crucial to understanding the biogeochemical behavior of Sb, given that sediments are an important sink for Sb in the environment. Such factors, however, have rarely been documented in natural sediments. In this study, we examined Sb species in 40 sediments of the world's largest Sb mine (Xikuangshan) located in southern China and tried to relate them to not only geochemical properties of the sediments but also their microbiological parameters obtained with the next‐generation DNA sequencing and metagenomics. We showed that Sb(III) accounted for <30% of inorganic Sb [Sb(III) + Sb(V)] in the sediments. Linear regression analysis revealed that the percentage of Sb(III) was negatively correlated to redox potential (Eh) but positively correlated to concentration of Fe(II), electrical conductivity, and the relative abundance of Sb(V)‐reducing microbes of the sediments, although the relative abundance of Sb(III)‐oxidizing microbes and the number of microbial genes responsible for Sb(III) oxidation were uncorrelated to the percentage of Sb(III). The observed strong associations indicated that Eh, concentration of Fe(II), electrical conductivity, and Sb(V)‐reducing microbes were involved in influencing Sb species in the sediments. On this basis, we further proposed a preliminary biogeochemical model for Sb speciation in water‐sediment systems affected by Sb‐mining activities. This study improved our understanding of the biogeochemical factors involved in influencing Sb species in natural sediments contaminated by Sb‐mining activities.