[Objective] Multifarious modular modifications in type I polyketide synthase (PKS) serve as crucial contributing factors for the diversity of polyketides. Ansamitocins, an antitumor agent, possess unique olefin shifts in the region of C11-C14, which might be catalyzed by dehydratase domain in PKS module 2 and 3. We evaluated the biochemical function of dehydratase domain in module 2 (Ansa DH₂). [Methods] Using ansamitocin-producing strain Actinosynnema pretiosum subsp. pretiosum ATCC 31280, we chose four different Ansa DHs in the ansamitocin biosynthetic modules to achieve bioinformatics analysis. Coupled with chemical synthesis of an analogue substrate waq-1 of Ansa DH₂, we presented our in vitro investigations. ESI-MS² analysis revealed the structure of the final product, which confirmed the α-β dehydration activity of Ansa DH₂. Ultimately, the correlation between the catalytic function and requisite amino acid residues has been determined through site-directed mutagenesis. [Results] Bioinformatics analysis suggests that Ansa DH₂ and Ansa DH₃ were closely related to those known DHs, which are responsible for olefin shift. An effective synthetic route was accomplished to afford waq-1 in 17.81% yield over six linear steps. The conversion rate of waq-1 reached 45% after 36 h by optimized in vitro enzymatic reactions. The α-β dehydration activity of Ansa DH₂ was reaffirmed by fragmentations in MS² profiles. Residue His48 in Ansa DH₂ was turned out to be indispensable for dehydration function by site-directed mutagenesis. [Conclusion] This work focused on the biochemical function of Ansa DH₂, which not only verified its α-β dehydration activity, but also shed light on further mechanistic study of olefin shift.