In this study, the mechanisms by which ferric iron promotes bioleaching of arsenopyrite by the moderately thermoacidophilic strain Sulfobacillus thermosulfidooxidans YN-22 was evaluated for the first time by integrating Fe L-edge and As/S K-edge X-ray absorption near edge structure (XANES) spectroscopy with Fourier transform infrared spectroscopy (FT-IR), synchrotron radiation X-ray diffraction (SR-XRD), Raman spectroscopy, and scanning electron microscopy (SEM), and the leaching parameters were also determined. The results showed that the addition of Fe3+ (6 g/L) significantly promoted bioleaching of arsenopyrite by enhancing the cell growth and the (bio)degradation of elemental sulfur (S-0) and orpiment (As2S3) intermediates on the mineral surface. In addition, an increased formation of loose particulate structures containing the Fe(III)-containing products jarosite (mainly ammoniojarosite), scorodite (ferric arsenate) and schwertmannite was observed.