Hop (Humulus lupulus L.) is central to beer brewing, particularly due to its α-bitter acids, which contribute to beer’s characteristic bitterness. Despite extensive research on their biosynthesis, the final oxidation step at the C6 position, determining the chiral configuration of α-bitter acids, remains unclear. In this study, we identify the flavin-dependent monooxygenase MO18 as the key enzyme catalyzing this oxidation, leading to the accumulation of (6S)-α-bitter acids in hop glandular trichomes. Co-expression of MO18 in yeast enhances (6S)-α-bitter acid production, while its absence results in a racemic mixture. MO18 interacts with a membrane-bound prenyltransferase, forming a metabolon that supports efficient biosynthesis. Thus, we designate MO18 as (6S)-α-bitter acid synthase ((6S)-α-BAS). Our work lays the groundwork for the future development of cost-effective, flavor-diverse craft beers.