This study aimed to reveal the specific mechanism of extracellular polymeric substances (EPS) in the bioleaching of copper-bearing sulfide ore by moderately thermophilic bacterium Acidithiobacillus caldus. The bioleaching performance of blank control (BC), planktonic cell deficient (PD), attached cell deficient (AD), and EPS deficient (ED) systems were compared, to investigate the specific functions of “non-contact” and “contact” (including direct contact and, EPS-mediated contact) mechanisms. The detailed mechanics of bioleaching were studied using μ_x of cell growth, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The μ_x of cell growth demonstrated that EPS favors planktonic and attached cell growth. SEM observation revealed that intensive micro-pores on slag benefitted from the “EPS-mediated contact” mechanism. XRD identification indicated that additional chemical derivatives were produced via “EPS-mediated contact” mechanism, because of the active iron/sulfur metabolism. FTIR analysis revealed that the absorption peaks of C-O-S, sulfate, and S = O, which are closely associated with sulfur metabolism, have significant influences of EPS secretion. Taken together, the “EPS-mediated contact” mechanism contributed to almost half of the “contact” mechanism efficiency and a quarter of the total bioleaching efficiency. The proposed specific mechanism of EPS can deepen our understanding of similar bioleaching processes.