Abstract: In order to investigate the changes of alkaloid metabolism during tobacco leaf senescence, the contents of different alkaloids in the middle leaves of Honghuadajingyuan were analyzed after topping. Meanwhile the expression of genes related to nicotine metabolism was analyzed based on RNA-seq data and further verified by Real-time PCR. The results suggested that the contents of alkaloids, including nicotine, nornicotine, anatabine and myosmine, increased gradually during leaf senescence. Many nicotine metabolism related genes were up-regulated during senescence, including JAT1, MATE2 and NUP1 which control nicotine transportation; CYP82E2 and CYP82E4 that are involved in nicotine transformation; nicotine synthesis genes ADC1, ODC2 and BBL2; COI1, JAZ1, MYC3 and MYC4 that are implicated in regulating nicotine metabolism. One of the regulating genes of nicotine metabolism, MTHFR1, on the other hand, was shown to be down-regulated during tobacco leaf senescence. These genes might co-regulate the biosynthesis and accumulation of nicotine in tobacco leaves.