Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. Their biosyntheses require two precursors, isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). There are two different pathways for the synthesis of IPP and DMAPP: the deoxyxylulose phosphate (DXP) pathway and the mevalonic acid (MVA) pathway. More importantly, these two pathways have a well-defined distribution among different kingdoms. Most pathogenic bacteria and protozoan parasites utilize the DXP pathway, while animals synthesize their isoprenoid precursors from acety-CoA via the MVA pathway. Plants have both DXP and MVA pathways. Thus, mechanistic studies on the DXP pathway enzymes may lead to the development of mechanism-based inhibitors as herbicides, broad-spectrum antibiotics, and antimalaria drugs. IspH in the DXP pathway catalyzes the reductive dehydration of (E)-4-hydroxy-3-metho-2-butenyl diphosphate (HMBPP) to form IPP and DMAPP, the last step in the DXP pathway. Recent EPR studies reported in literature suggest that IspH is a unique iron-site-containing [4Fe-4S] protein. In this study, we studied the IspH-catalyzed reductive dehydration mechanism using two substrate analogues. Our data reported herein provide evidence to not only support the integrity of the C1 position C-O bond during reaction, they also suggest a heterolytic C-O bond cleavage at the C4 position for IspH-catatyzed reductive dehydration reaction. Our kinetic studies also suggest that the C4 hydroxyl group is involved in substrate binding. Because the IspH-catalyzed reductive dehydration reaction does not fall into the two known classes of unique iron-site-containing [4Fe-4S] proteins, aconitase-type and radical SAM-type enzymes, IspH may represent a new class of iron-sulfur-containing proteins.