Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop. •In the present study, we generated Hlmed5a/med5b double mutant line using CRISPR/Cas9 technique. The Hlmed5a/med5b double mutant line exhibited reduction in secondary metabolite content.•In addition, we observed the robust growth, earlier floral, cone maturation and distortion in lupulin gland morphology in Hlmed5a/med5b double mutant line.•The comparative transcriptome analysis unveiled the modulation of global gene expression patterns in Hlmed5a/med5b double mutant line related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development.