Iron superoxide dismutase (Fe-SOD) is predominantly found in bacteria and mitochondria. The thermal stability of Fe-SOD from different sources can vary dramatically. We have studied the influence of structural parameters on Fe-SOD thermostability by principal component analysis (PCA). The results show that an increased α-helical and turn content, an increased α-helix and loop length, an increase in the number of main-main chains and charged-uncharged hydrogen bonds, a decrease in the 310-helix content, and a decreased β-strand and loop length are all important factors for Fe-SOD thermostability. Interestingly, the use of charged residues to form salt bridges is tendentious in thermophilic Fe-SOD. Negatively charged Arg and positively charged Glu are efficiently used to form salt bridges. The cooperative action of the exposed area, the hydrogen bonds, and the secondary structure plays a crucial role in resisting high temperatures, which demonstrates that the increased stability of thermophilic Fe-SOD is provided by several structural factors acting together. © 2012 Wiley Periodicals, Inc. Biopolymers 97:864–872, 2012.