Glycosylation determines the complexity and diversity of plant natural products. To characterize fruit ripening-related UDP-dependent glycosyltransferases (UGTs) functionally in strawberry, we mined the publicly available Fragaria vesca genome sequence and found 199 putative UGT genes. Candidate UGTs whose expression levels were strongly up-regulated during fruit ripening were cloned from F.×ananassa and six were successfully expressed in Escherichia coli and biochemically characterized. UGT75T1 showed very strict substrate specificity and glucosylated only galangin out of 33 compounds. The other recombinant enzymes exhibited broad substrate tolerance, accepting numerous flavonoids, hydroxycoumarins, naphthols and the plant hormone, (+)-S-abscisic acid (ABA). UGT71W2 showed the highest activity towards 1-naphthol, while UGT71A33, UGT71A34a/b and UGT71A35 preferred 3-hydroxycoumarin and formed 3- and 7-O-glucosides as well as a diglucoside from flavonols. Screening of a strawberry physiological aglycone library identified kaempferol, quercetin, ABA and three unknown natural compounds as putative in planta substrates of UGT71A33, UGT71A34a and UGT71W2. Metabolite analyses of RNA interference (RNAi)-mediated silenced fruits demonstrated that UGT71W2 contributes to the glycosylation of flavonols, xenobiotics and, to a minor extent, of ABA, in planta. The study showed that both specialist and generalist UGTs were expressed during strawberry fruit ripening and the latter were probably not restricted to only one function in plants.