Prenylation usually improves structural diversity and bioactivity in natural products. Unlike the discovered enzymatic gem-diprenylation of mono- and tri-cyclic aromatic systems, the enzymatic approach for gem-diprenylation of bi-cyclic hydroxynaphthalenes is new to science. Here we report an enzymatic example for dearomative C4 gem-diprenylation of α-hydroxynaphthalenes, by the F253G mutant of a fungal prenyltransferase CdpC3PT. Experimental evidence suggests a sequential electrophilic substitution mechanism. We also explained the alteration of catalytic properties on CdpC3PT after mutation on F253 by modeling. This study provides a valuable addition to the synthetic toolkit for compound prenylation and it also contributes to the mechanistic study of prenylating enzymes.