We isolated a cDNA encoding S-adenosylmethionine: anthocyanin 3′,5′-O-methyltransferase (A3′5′OMT) from a cDNA library derived from Torenia hybrida petals that mainly accumulated malvidin type anthocyanins using the petunia A3′OMT cDNA as a probe. The torenia A3′5′OMT shared 52–72% amino acid sequence identity with previously reported AOMTs and belongs to the Group A1 methyltransferase family that also include caffeoyl CoA O-methyltransferase. The recombinant A3′5′OMT produced by Escherichia coli efficiently catalyzed methylation of the 3-glucoside and 3,5-diglucoside of delphinidin and cyanidin, but it did not catalyze the methylation of anthocyanidins, flavonols, or flavones. The torenia A3′5′OMT gene was expressed in Nierembergia sp., the petals of which naturally accumulate anthocyanins derived from delphinidin. The resultant transgenic petals produced methylated anthocyanins, based on malvidin and petunidin, in addition to delphinidin, which indicated that the torenia A3′5′OMT gene was functional in a heterologous plant. Rose petals rarely contain methylated anthocyanins. Transgenic rose petals expressing both a pansy flavonoid 3′,5′-hydroxylase (F3′5′H) and the torenia A3′5′OMT genes accumulated methylated anthocyanins based upon malvidin, petunidin, and peonidin, which comprised up to 88% of the total anthocyanidins, and their magenta color was more brilliant than that of the petals that accumulated delphinidin type anthocyanins by expressing the F3′5′H gene alone. These results indicate that the torenia A3′5′OMT gene is a useful molecular tool for altering and diversifying flower color.