The relative configuration of natural roflamycoin (1) was determined by C-13 acetonide analysis, combined with other supporting spectroscopic data. A previous proposal for the configuration of roflamycoin was shown to be incorrect. The absolute configuration was determined by the advanced Mosher’s method. Our proposed structure of natural roflamycoin was confirmed by the synthesis of a degradation fragment that incorporates all of the 11 independent stereogenic centers of roflamycoin. The complete structure of roflamycoin is illustrated in Figure 1.