Extremophiles display an astonishing array of adaptations to harsh environmental conditions. We analyzed the mechanisms of ionizing radiation resistance from a diverse group of extremophilic archaea and bacteria. In Halobacterium salinarum IR resistance is conferred by antioxidant Mn2+-complexes, and protein-free cell extracts (ultrafiltrates, UFs) of super-resistant (IR+) isolates of H. salinarum had increased concentrations of Mn, PO4 and amino acids compared to the founder strain. Proteomic analysis determined that IR+ isolates with increased Mn had elevated protein expression for central carbon metabolism, suggesting a Mn-stimulated metabolic route to increased IR resistance. We examined the role of mannosylglycerate, di-myo-inositol phosphate, and trehalose in the IR resistance of various thermophiles; aerobic thermophiles had UFs which were radioprotective of enzyme activity under aerobic conditions, which is attributed to Mn, PO4 and trehalose accumulation. In contrast, anaerobic thermophile UFs did not contain significant amounts of Mn, and were radioprotective only under anaerobic conditions; we conclude the anaerobic environment confers their IR resistance.