Anaerobic microbiological antimonate [Sb(V)] respiration is a newly discovered process regulating the Sb redox transformation in soils. However, little is known about the role microbiological Sb(V) respiration plays in the fate of Sb in the subsurface, especially in the presence of sulfate and electron shuttles. Herein, we successfully enriched a Sb(V) reducing microbiota (SbRM) from the subsurface near an active Sb mine. SbRM was dominated by genus Alkaliphilus (18-36%), Clostridiaceae (17-18%), Tissierella (24-27%), and Lysinibacillus (16-37%). The incubation results showed that SbRM reduced 88% of dissolved Sb(V) to Sb(III), but the total Sb mobility remained the same as in the abiotic control, indicating that SbRM alone did not increase the total Sb release but regulated the Sb speciation in the subsurface. Micro X-ray fluorescence (μ-XRF) analysis suggested the association of Sb and Fe, and electron shuttles such as anthraquinone-2,6-disulfonic disodium salt (AQDS) markedly enhanced the Sb release due to its ability to facilitate Fe mineral dissolution. Sb L-edge and S K-edge X-ray absorption near edge structure (XANES) results demonstrated that indigenous SbRM immobilized Sb via Sb2S3 formation, especially in a sulfur-rich environment. The insights gained from this study shed new light on Sb mobilization and its risk assessment in the subsurface environment.