Lactarius volemus is a mushroom species that produces beneficial organic compounds. A large number of these compounds are isoprenoids. Isopenteyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are essential for isoprenoid biosynthesis. The reversible isomerization of IPP to DMAPP is catalyzed by isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2). However, there is no information on IDI from mushrooms. In this study, we report a novel cDNA encoding IDI from L. volemus (LvIDI). LvIDI was composed of 253 amino acid residues with a molecular weight of 28.8 kDa and anisoelectric point of 4.99. The kinetic assay results indicated that the Km was 0.39 μM and Vmax was 0.034 μmol mg-1 min-1. Moreover, LvIDI was classified into Basidiomycota in the phylogenetic analysis.