In previous work we reported the formal synthesis of the marine macrolide (−)-callyspongiolide through a synthetic approach in which the generation of the key macrocyclic core relies on a ruthenium-catalyzed ring-closing metathesis reaction. However, besides the predicted macrolactone intermediate, an undesired cyclooctene was observed. We investigate here the mechanism of this critical step through density functional theory calculations. The results indicate that the chemoselectivity is not under kinetic control but ruled by thermodynamics. This conclusion is further confirmed by additional experimental studies.