Anthocyanins are a group of colorful and bioactive natural pigments with important physiological and ecological functions in plants. We found an MYB transcription factor (PtrMYB119) from Populus trichocarpa that positively regulates anthocyanin production when expressed under the control of the CaMV 35S promoter in transgenic Arabidopsis. Amino acid sequence analysis revealed that PtrMYB119 is highly homologous to Arabidopsis PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), a well-known transcriptional activator of anthocyanin biosynthesis. Independently produced transgenic poplars overexpressing PtrMYB119 or PtrMYB120 (a paralogous gene to PtrMYB119) (i.e., 35S::PtrMYB119 and 35S::PtrMYB120, respectively) showed elevated accumulation of anthocyanins in the whole plants, including leaf, stem and even root tissues. Using a reverse-phase high-performance liquid chromatography, we confirmed that the majority of the accumulated anthocyanin in our transgenic poplar is cyanidin-3-O-glucoside. Gene expression analyses revealed that most of the genes involved in the anthocyanin biosynthetic pathway were highly upregulated in 35S::PtrMYB119 poplars compared with the nontransformed control poplar. Among these genes, expression of PtrCHS1 (Chalcone Synthase1) and PtrANS2 (Anthocyanin Synthase2), which catalyze the initial and last steps of anthocyanin biosynthesis, respectively, was upregulated by up to 350-fold. Subsequent transient activation assays confirmed that PtrMYB119 activated the transcription of both PtrCHS1 and PtrANS2. Interestingly, expression of MYB182, a repressor of both anthocyanin and proanthocyanidin (PA) biosynthesis, was largely suppressed in 35S::PtrMYB119 poplars, while expression of MYB134, an activator of PA biosynthesis, was not changed significantly. More interestingly, high-level accumulation of anthocyanins in 35S::PtrMYB119 poplars did not have an adverse effect on plant growth. Taken together, our results demonstrate that PtrMYB119 and PtrMYB120 function as transcriptional activators of anthocyanin accumulation in both Arabidopsis and poplar.