The evolutionary adaptations of functional genes to life at high pressure are not well understood. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the myosin heavy chain (MyHC) cDNA from skeletal muscle of two deep-sea fishes, Coryphaenoides yaquinae and C. armatus, and two non-deep-sea fishes, C. acrolepis and C. cinereus. The MyHCs of deep-sea fishes have a unique structure in two loop regions, loop-1 and loop-2, in comparison with those of non-deep-sea fishes. The loop-1 region of deep-sea fishes has a Pro residue and the loop-2 region, which is an actin-binding site, is shorter than the same region in non-deep-sea fishes. The amino acid substitution in the loop-1 region is expected to be mainly involved in ATPase activity, whereas the deletion in the loop-2 region affects the association of MyHC with actin filaments at high pressure. In addition, the MyHC of deep-sea fishes has biased amino acid substitutions at core positions in the coiled-coil structure of the rod region. These amino acid substitutions are likely to decrease the cavities in the coiled-coil structure and consequently make the structure more compact and unaffected by high pressure. Together, these results indicate that amino acid substitutions can adaptively alter the pressure sensitivity of a protein even if they do not directly influence core structure.