[Background] Skyllamycins are nonribosomal cyclic depsipeptides that possess inhibitory activity against the platelet-derived growth factor (PDGF) signaling pathway and antibiofilm activity. The cyclization reaction in skyllamycin biosynthesis is catalyzed by the thioesterase domain. [Objective] To characterize the substrate promiscuity of thioesterase domain, we cloned and expressed the thioesterase-encoding gene, synthesized substrate mimics and carried out the cyclization reactions in vitro. [Methods] The thioesterase domain (Skyxy-TE)-encoding gene was cloned from Streptomyces sp. PKU-MA01239 with ligation-independent cloning method, Skyxy-TE was purified by Ni-NTA affinity chromatography, and substrate mimics were synthesized by using solid-phase peptide synthesis (SPPS). [Results] Soluble Skyxy-TE was obtained and purified to homogeneity, and two substrate mimics 1 and 2 were synthesized by SPPS. Cyclization reactions were carried out in vitro, leading to the production of two cyclized peptides 3 and 4, whose structures were elucidated by NMR and HRESIMS analysis. [Conclusion] Skyxy-TE showed substrate promiscuity by catalyzing two substrate mimics to generate cyclization products, which facilitates the generation of more cyclized peptide analogues by chemoenzymatic synthesis in the future.