Abstract: Vanadium and its derivatives are used in various industries, including steel, metallurgy, pharmaceuticals, and aerospace engineering. Although China has massive reserves of stone coal resources, these resources have low grades. Therefore, the effective extraction and recovery of metallic vanadium from stone coal is an important way to realize the efficient resource utilization of stone coal vanadium ore. Herein, Bacillus mucilaginosus was selected as the leaching strain. The vanadium leaching rate reached 35.5% after 20 d of bioleaching under optimal operating conditions. The cumulative vanadium leaching rate in the contact group reached 35.5%, which was higher than that in the noncontact group (9.3%). The metabolites of B. mucilaginosus, such as oxalic, tartaric, citric, and malic acids, dominated in bioleaching, accounting for 73.8% of the vanadium leaching rate. Interestingly, during leaching, the presence of stone coal stimulated the expression of carbonic anhydrase in bacterial cells, and enzyme activity increased by 1.335–1.905 U. Enzyme activity positively promoted the production of metabolite organic acids, and total organic acid content increased by 39.31 mg·L⁻¹, resulting in a reduction of 2.51 in the pH of the leaching system with stone coal. This effect favored the leaching of vanadium from stone coal. Atomic force microscopy illustrated that bacterial leaching exacerbated corrosion on the surface of stone coal beyond 10 nm. Our study provides a clear and promising strategy for exploring the bioleaching mechanism from the perspective of microbial enzyme activity and metabolites.