It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.