Imperata cylindrica is known to produce a pair of triterpenes, isoarborinol and fernenol, that exhibit identical planar structures but possess opposite stereochemistry at six of the nine chiral centers. These differences arise from a boat or a chair cyclization of the B-ring of the substrate. Herein, we report the characterization of three OSC genes from I. cylindrica. IcOSC1 and IcOSC5 were identified as isoarborinol and fernenol synthases, respectively, while IcOSC3 was characterized as a multifunctional enzyme that produces glutinol and friedelin as its major products. Mutational studies of isoarborinol and fernenol synthases revealed that the residues surrounding the DCTAE motif partially affected the conformation of the B-ring during cyclization. Additionally, the IcOSC1-W255H mutant produced the rare triterpene boehmerol. The introduced histidine residue presumably abstracted a proton from the intermediary carbocation at C18 during the 1,2-rearrangement. Expression analysis indicated that all OSC genes were highly expressed in stems.