Chlorophyll is responsible for absorbing light which is required to convert solar energy into chemical energy during photosynthesis. Despite remarkable progresses achieved in chlorophyll biosynthesis, the relevant mechanisms in poplar remain unclear. Here, we identified a BBX gene, PabBBX27, which was from Populus alba × P. Berolinensis, may regulate the biosynthesis of chlorophyll in poplar. PabBBX27 transcripts were most abundant in young leaves of poplar. Transgenic lines overexpressing (OE) PabBBX27 were characterized by dark green leaf. The contents of chlorophyll and photosynthates in the OE lines were significantly increased. In addition, photosynthetic rate, stomatal guidance, intercellular CO₂ concentration, transpiration rate, initial fluorescence F₀, maximum light efficiency (Fv/Fm), actual photochemical quantum yield and photochemical quenching of the OE lines were all increased. Genes relate to chlorophyll metabolic pathway and photosynthesis were activated in the OE lines as well. Yeast one-hybrid results showed that PabBBX27 may enhance their activities by binding to the G-box elements in the promoter sequences of these genes. Taken together, our results provide a new insight for the molecular mechanism of chlorophyll metabolic pathway and the cultivation of new varieties with high photosynthetic utilization efficiency for poplar.