The enzymatic cyclization reaction of polyprenoid C(33) by squalene-hopene cyclase (SHC) was investigated with the intention of creating an unnatural hexacyclic compound. The enzymatic products consisted of mono-, bi-, tri-, tetra- and pentacyclic skeletons; however, hexacyclic products were not generated, contrary to our expectations. The absence of a hexacyclic skeleton indicated that the entire carbon chain of C(33) polyprene could not be included in the reaction cavity. Formation mechanisms of the products having mono- to pentacycles were discussed. Both chair/chair/boat conformation and chair/chair/chair conformations were formed for a tricycle, and both chair/chair/chair/boat conformation and chair/chair/chair/chair structures were constructed for a tetracycle. The pentacyclic product was created from the chair/chair/chair/chair/boat conformation. Squalene was folded in an all pre-chair conformation inside the reaction cavity to form the hopene skeleton. Therefore, the formation of a boat structure during the polycyclization reaction indicated that the molecule of polyprene C(33) was folded improperly due to incorrect arrangement/positioning in the reaction cavity. The creation of the hexacyclic core failed; however, it should be noted that SHC possessed great potential to tolerate the elongated squalene analog C(33), thus leading to the creation of novel compounds with C(33).