d‐Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold‐type III pyridoxal 5′‐phosphate‐dependent enzyme catalyzing d‐serine dehydration. The enzyme contains 1 mol Zn2+ in its active site and shows a unique zinc dependence. The Zn2+ is essential for the d‐serine dehydration, but not for the α,β‐elimination of β‐Cl‐d‐alanine catalyzed as a side‐reaction. The fact that dehydration of d‐threonine and d‐allo‐threonine, also catalyzed by DsdSC, is likewise Zn2+ dependent indicates that Zn2+ is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of Cβ. Removal of Zn2+ results in a less polar active site without changing the gross conformation of DsdSC. 1H NMR determined the rates of α‐hydrogen abstraction and hydroxyl group elimination of d‐serine in 2H2O to be 9.7 and 8.5 s−1, respectively, while the removal of Zn2+ abolished both reactions. Mutation of Cys400 or His398 within the Zn2+ binding sites to Ala endowed DsdSC with similar properties to those of the Zn2+‐depleted wild‐type enzyme: the mutants lost the reactivity toward d‐serine and d‐threonine but retained that toward β‐Cl‐d‐alanine. 1H NMR analysis also revealed that both α‐hydrogen abstraction and hydroxyl group elimination from d‐serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α‐hydrogen abstraction and hydroxyl group elimination in a concerted fashion.