Objectives To produce flavonol and flavone 6-C-glucosides by bioconversion using recombinant Escherichia coli expressing a C-glucosyltransferase from wasabi (WjGT1). Results Escherichia coli expressing WjGT1 (Ec-WjGT1) converted flavones (apigenin and luteolin) and flavonols (quercetin and kaempferol) into their 6-C-glucosides in M9 minimal media supplemented with glucose, and released these products into the culture media. Ec-WjGT1 system also converts a flavanone (naringenin) into its C-glucoside at a conversion rate of 60% in 6 h. For scale-up production, apigenin, kaempferol, and quercetin were sequentially fed into the Ec-WjGT1 system at concentrations of 20-50 mu M every 15-60 min, and the system was then able to produce isovitexin, kaempferol 6-C-glucoside, and quercetin 6-C-glucoside at an 89-99% conversion rate. Conclusions The Ec-WjGT1 system quickly and easily produces flavone and flavonol 6-C-glucosides at high conversion rates when using sequential administration to avoid precipitation of substrates.