Ring formation is a common strategy to diversify polyketide natural products that requires specific folding of the polyketide chain, but precisely how polyketide synthase (PKS) accomplishes this feat of origami is a mystery. Presented herein are the crystal structures and enzymological studies of several PKS domains related to cyclization specificity: the ketoreductase (KR) and the aromatase/cyclase (ARO/CYC) in type II PKS, the product template (PT) and thioesterase (TE) domains in nonreductive fungal PKS, and domains involved in reductive fungal polyketide biosynthesis. Despite low sequence homology, several domains from different types of PKS and fatty acid synthase (FAS) have a striking similarity in their domain architecture and substrate binding pocket, while the different cavity size and shape is closely related to the distinct cyclization specificity observed in each domain. The sequence-structure-function relationship that results in the unique regio-specificity of each PKS will be discussed in details.