Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase III clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum (EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4'-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria (higher pH than cytoplasm) of the 8-prenylkaempferol-producing yeast strain or co-culturing the 8-prenylkaempferol-producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.