Zizaene synthase (ZS) from Chrysopogon zizanioides (Poaceae) is the critical enzyme in the biosynthesis of the fragrant sesquiterpene khusimol, a major component of the vetiver essential oil used widely by the cosmetic industry. As reported previously, we heterologously and successfully ex-pressed the active ZS with a small ubiquitin-related modifier (SUMO) fusion domain. In this study, we report the optimization of reaction conditions and determination of enzyme kinetics of ZS. Moreover, we investigate the catalytic specificity and reaction mechanisms with the ubiquitous (2E,6E)-farnesyl diphosphate (FDP) and with C10 and C15 prenyl diphosphate isomers. Catalytic promiscuity occurs with monoterpene substrates generating eight products that comprise acyclic, cyclic, and hydroxylated monoterpenes. In contrast, ZS is a high-fidelity terpene cyclase when used with C15 isomer substrates, yielding as major products (Z)-β-farnesene (100%) for (2E,6Z)-FDP and (+)-zizaene (81.7%), β-acoradiene (12.8%), and (E)-β-farnesene (5.5%) for (2Z,6E)-FDP. Cyclization of the ubiquitous substrate (2E,6E)-FDP demonstrates a higher catalytic specificity, whereas the reaction proceeds via the acorenyl cation that generates (+)-zizaene (91.5%) and β-acoradiene (8.5%). Furthermore, catalytic specificity with (2E,6E)-FDP was stable in reactions tested at distinct pH and temperatures, suggesting a stable and efficient closed conformation of the active site during catalysis. To understand such stability, open and closed structural conformations of ZS were modeled in silico and revealed putative residues in the active site and in the A-C and J-K surrounding loops, which could explain the high fidelity of ZS.