This work describes a chiral bifunctional squaramide/DBU sequential catalytic strategy for the enantioselective synthesis of nonfused chiral eight-membered O-heterocycles through the asymmetric addition of ynones to β,γ-unsaturated α-ketoesters followed by the regio- and diastereoselective cyclization of the adduct intermediates. Mechanistic experiments revealed that an isomerization process should be involved in the ring formation step, and the origin of the high regioselectivity and diastereoselectivity has also been elucidated by the DFT calculations.