Anthocyanin is the primary pigment contributing to red, violet, and blue flower color formation. The solubility of anthocyanins is enhanced by UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT) through transfer of the glucosyl moiety from UDP-glucose to 3-hydroxyl group to produce the first stable pigments. To assess the possibility that UFGT is involved in the flower color formation in Phalaenopsis, the transcriptional activities of PeUFGT3, and other flower color-related genes in developing red or white flower buds were examined using RT-PCR analysis. In contrast with chalcone synthase, chalcone isomerase, and anthocyanidin synthase genes, PeUFGT3 transcriptional activity was higher expressed in the red color of Phalaenopsis cultivars. In the red labellum of Phalaenopsis 'Luchia Lady', PeUFGT3 also showed higher expression levels than that in the white perianth. PeUFGT3 was predominantly expressed in the red region of flower among various Phalaenopsis cultivars. To investigate the role of PeUFGT3 in red flower color formation, PeUFGT3 was specifically knocked down using RNA interference technology via virus inducing gene silencing in Phalaenopsis. The PeUFGT3-suppressed Phalaenopsis exhibited various levels of flower color fading that was well correlated with the extent of reduced level of PeUFGT3 transcriptional activity. Furthermore, there was a significant decrease in anthocyanin content in the PeUFGT3-suppressed Phalaenopsis flowers. The decrease of anthocyanin content due to PeUFGT3 gene silencing possibly caused the faded flower color in PeUFGT3-suppressed Phalaenopsis. Consequently, these results suggested that the glycosylation-related gene PeUFGT3 plays a critical role in red color formation in Phalaenopsis.