Variotin, an antifungal antibiotic produced by Paecilomyces varioti Bainier var. antibioticus with the molecular formula C17H28O5N, was previously proposed to have the structure N-(8'-R-hydroxy-6'-methyl-trans-trans-cis-dodeca-2',4',6'-trienoyl)-2-pyrrolidone, with the cis configuration of the 6' double bond inferred from a Diels-Alder reaction. This study utilized the intramolecular nuclear Overhauser effect (NOE) in NMR to re-determine the configuration of the 6' double bond. NMR spectra were recorded at 100 MHz in deuterated benzene at a concentration of 100 mcg/ml. Irradiation of the 13' allylic methyl signal at 1.65 ppm from TMS resulted in a ~12% increase in the integrated intensity of the quartet signal due to the 8' proton and a ~9% increase in the total area of signals due to the 4' and overlapping 5' olefinic protons at 6.4 ppm, while the integrated area of the 1' doublet at 3.1 ppm was insignificant. From these NOE results, it was concluded that the 8' proton is closer to the 13' methyl group than the 1' proton, indicating the 6' double bond has a trans configuration. This contradicts the previous chemical conclusion from the Diels-Alder reaction (attributed to possible stereochemical change during the reaction), leading to the revised structure of variotin as N-(8'-R-hydroxy-6'-methyl-trans-trans-trans-dodeca-2',4',6'-trienoyl)-2-pyrrolidone.