Abstract: Chalcone isomerase (CHI) is one of the key enzymes in flavonoid metabolism. In tobacco, two copies of CHI genes, NtCHI1 and NtCHI2, are identified, while little is known on the function of NtCHI2 compared to that of NtCHI1. To study the function of NtCHI2, its rutin content, CHI activity, and NtCHI2 expression level were analyzed for NtCHI2-OE strains and NtCHI2-RNAi strains. We also examined the CHI activity of recombinant NtCHI2 expressed in vitro. The results showed that NtCHI2 had a CHI activity. In the NtCHI2-OE strains, the NtCHI2 expression level was up-regulated by 5-75 folds, however no significant changes in CHI activity and rutin content were detected. In NtCHI2-RNAi strains, the NtCHI2 expression level was down-regulated to 70%, and correspondingly, CHI activity and rutin content decreased by 50% and 90% respectively. NtCHI2 was involved in the metabolic regulation of rutin, and the expression of NtCHI2 was positively correlated with the rutin content to some extent.