Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.