This study examined the microbial reduction of hexavalent chromium [Cr(VI)] by an extremely thermophilic bacterium, Caldicellulosiruptor saccharolyticus, under glucose fermentation conditions at 70 °C. Experimentation with different initial Cr(VI) concentrations confirmed that C. saccharolyticus had the ability to reduce Cr(VI) and immobilize Cr(III). At a concentration of 40 mg/L, Cr(VI) was completely reduced within 12 h, and 97% of the reduction product Cr(III) precipitated on the cell surface. Cr(VI) reduction was accelerated by the addition of neutral red (NR, an electron mediator), resulting in the reduction time shortened to 1 h. The addition of CuCl2, a Ni-Fe hydrogenase inhibitor, also enhanced Cr(VI) reduction. Additionally, analysis of the relationship between Cr(VI) reduction and glucose fermentation suggested that different electron sources acted during CuCl2 and NR conditions. Hydrogen served as an electron donor under normal fermentation and NR conditions with the catalysis of Ni-Fe hydrogenase. However, when the activity of Ni-Fe hydrogenase was inhibited by CuCl2, C. saccharolyticus directly used reduction equivalents during glucose fermentation for intracellular Cr(VI) reduction. Therefore, our findings demonstrated high Cr(VI) reduction ability and different electron transfer pathways during Cr(VI) reduction by C. saccharolyticus.