Plant polyphenols play important roles in defense against pathogens, promoting production quality, and improving human health. Light is an important external environmental factor that greatly impacts on the polyphenols metabolism, whereas the impact mechanism remains unknown. In order to investigate whether light receptors participate in the regulation of polyphenols metabolism in tobacco (Nicotiana tabacum) leaf, a Phytochrome B homolog, NtPHYB1, was isolated from N. tabacum cv. K326, and its function on polyphenols metabolism was carried out by over expression and RNAi approaches. Consistent and complemental results indicated that NtPHYB1 is involved in polyphenols metabolism in tobacco leaves, and the high level of NtPHYB1 transcripts is in favour of accumulation of chlorogenic acid and its isomers, all of which are key components of tobacco polyphenols. To understand the regulation mechanism of NtPHYB1 in the secondary metabolism pathway, transcriptome analysis was carried out. Compared with WT, 1665 and 1421 genes are found differentially-expressed in NtPHYB1-GFP and NtPHYB1-RNAi transgenic lines respectively. Among them, about 30 genes were related to phenylpropanoid biosynthesis, which is the pathway for polyphenols biosynthesis. Further evidence from quantitative RT-PCR confirmed that NtPHYB1 may control polyphenol metabolism through regulating the transcription of PAL4 (phenylalanine ammonialyase 4), 4CL1 (4-coumarate: coenzyme A ligase 1) and COMT (caffeic acid 3-O-methyltransferase) genes. Our results indicated that phytochrome B is involved in regulating polyphenol metabolism in tobacco leaves. This perhaps provides a novel clue on the regulation mechanism and a strategy to improve the polyphenols accumulation.