Myxobacteria are efficient producers of numerous secondary metabolites, and the genus Sorangium is frequently described as a proficient source for new, biologically active natural products. We report here the discovery and complete structure elucidation of pellasoren from the myxobacterium Sorangium cellulosum. Pellasoren exhibits cytotoxicity against HCT-116 human colon cancer cells, with pellasoren A (all-E C10–C11 double bond) having an IC50 of 155 nm and pellasoren B (Z-configured at C10–C11) being approximately one magnitude less active (IC50 2.35 μm). Structural elucidation by NMR and ESI-MS confirmed features including an unusual enol ether moiety and a lactone moiety. Identification of the corresponding pel gene cluster from S. cellulosum So ce38 allowed us to establish a model for pellasoren biosynthesis, providing evidence for an unusual route to glycolate extender unit generation. Moreover, we present the first total synthesis of pellasoren—using key steps such as Wittig olefination and stereoselective intramolecular protonation—which confirmed the absolute configuration of this natural product.