Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type‐I polyketide synthase, pimTE controls the 26‐m‐r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6‐m‐r hemiketal formation was linked to pimTE‐catalyzed 26‐m‐r lactonization. Compared to non‐hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U‐turn motif in the acyl‐enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6‐m‐r hemiketal. Moreover, the small‐model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6‐m‐r hemiketal cyclization. In addition, the post‐hemiketal molecular dynamic simulations demonstrate that hydrogen‐bonding networks surrounding the substrate U‐turn of the hairpin‐shaped conformation changes significantly when the 6‐m‐r hemiketal is formed. In particular, the R‐hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26‐m‐r lactonizing pre‐reaction state. By contrast, the S‐hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi‐functional enzyme, which can synergistically catalyze tandem 6‐m‐r and 26‐m‐r formations during the main‐chain release of pimaricin biosynthesis. [ABSTRACT FROM AUTHOR]