High-potential iron-sulfur proteins (HiPIPs) are an important class of metalloproteins with a [4Fe-4S] cluster coordinated by four cysteine residues. Distinct from other iron-sulfur proteins, the cluster in HiPIP has a high reduction potential, making it an essential electron carrier in bacterial photosynthesis. Here, we combined single-molecule atomic force microscopy and protein engineering techniques to investigate the mechanical unfolding mechanism of HiPIP from Chromatium tepidum (cHiPIP). We found that cHiPIP unfolds in a two-step fashion with the protein sequence sequestered by the iron-sulfur center as a stable unfolding intermediate state. The rupture of the iron-sulfur center of cHiPIP proceeds in two distinct parallel pathways; one pathway involves the concurrent rupture of multiple iron thiolate bonds, and the other one involves the sequential rupture of the iron-thiolate bonds. This mechanistic information was further confirmed by mutational studies. We found that the rupture of the iron-thiolate bonds in reduced and oxidized cHiPIP occurred in the range of 150-180 pN at a pulling speed of 400 nm/s, similar to that measured for iron-thiolate bonds in rubredoxin and ferredoxin. Our results may have important implications for understanding the general unfolding mechanism governing iron-sulfur proteins, as well as the mechanism governing the mechanical rupture of the iron-sulfur center.