Single-chain insulin analogs (SCIs) provide insight into the interrelation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (< 3 residues) prevent induced fit on receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, chemical synthesis, and solution structure of an active single-chain insulin analog (SCI-57) containing a six-residue linker (GGGPRR). Native receptor-binding affinity (129 (±8) percent relative to wild-type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of the SCI is markedly increased (Gu 0.7(±0.1) kcal/mole relative to the corresponding two-chain analog and 1.9(±0.1) kcal/mole relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects (NOEs) demonstrates that a nativelike fold is maintained. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core whereas the final Arg extends the A1-A8 -helix. Comparison between the SCI-57 and its parent two-chain analog reveals striking enhancement of multiple nativelike NOEs within the SCI-57. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultra-stable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.