Abstract Conformations of 14-membered macrolide—pikromycin, narbomycin, and their derivatives—in solution are studied based upon their NMR and CD spectra compared with the conformation of p-bromobenzoylpikromycin in solid state. The α,β-unsaturated CO group at the C-9 position in pikromycin, acetylpikromycin, p-bromobenzoylpikromycin, diacetylpikromycin, pikronolide, narbomycin and narbonolide has an s-trans conformation in solution of the protic solvent. While, in pikromycin, acetyl pikromycin, and p-bromobenzoylpikromycin, it has an s-cis conformation in solution of the aprotic solvent. This phenomenon has not been reported previously in macrolides, and is caused by an intramolecular H-bond between the OH group at the 12-position and the C-3 CO group in the aprotic solvent. This was confirmed from NMR, IR, and CD spectra. The conformation of the 14-membered macrolactone is discussed with reference to “diamond lattice” conformation models A, B, C. D, and F. A new “diamond lattice” conformation model E for pikromycin in the aprotic solvents is proposed based on spectral data.