Bitter acids (α-type and β-type) account for more than 30% of the fresh weight of hop (Humulus lupulus L.) glandular trichomes and are well-known for their contribution to the bitter taste of beer. These multi-prenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast system for the functional identification of aromatic prenyltransferase genes (PT). Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific cDNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Step-wise mutation of the Asp-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L only catalyzed the first prenylation step; PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two hybrid system, reciprocal co-immunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.