C-Glucosides are glucose-containing glycosides that have carbon–carbon bonds between the anomeric carbon of the sugar moieties and aglycon, rendering the molecules remarkably stable against hydrolysis by enzymes or acids. In this work, we showed the production of C-glucosides of flavonoids and related compounds (i.e., 2-hydroxyflavanone, dihydrochalcone, and trihydroxyacetophenone) by Escherichia coli expressing buckwheat C-glucosyltransferase. The substrates in their respective cultures were taken up by the cells and C-glucosylated, and the products were released into the culture media. The bioconversion process was completed in 1–2 h, but products were already observed immediately after addition of the substrates (200 µM). The conversion rates of these substrates reached 80–95%. Without addition of glucose to the conversion media, almost no C-glucosides were produced. Although the amounts of the substrates fed to their respective cultures were limited by their solubility in water, repeated addition of the substrate to the culture at regular time intervals effectively increased the total amount of product obtained.