Abstract: Carotenoid content in mature leaves is important for tobacco quality. In order to explore the changes of carotenoid components and related gene expression during tobacco leaf senescence, the contents of neoxanthin, lutein and β-carotene in the middle leaves of Honghuadajinyuan were analyzed in leaves from different developmental stages using liquid chromatography. Meanwhile, the expression of genes related to carotenoid metabolism was analyzed based on transcriptome data and quantitative real-time PCR verification. The results showed that the contents of neoxanthin, lutein, and β-carotene in tobacco leaves gradually decreased during tobacco leaf senescence and the carotenoid synthesis genes GGPS, PSY, PDS, ZDS, CRTISO, LYCB and LYCE were down-regulated. The carotenoid-degrading genes LOX, POD and CCD1 were up-regulated and PAL and PPO were down-regulated. The carotenoid converting genes ZEP, NXS and NCED were up-regulated. The transcription factors involved in the carotenoid synthesis process ORANGE, HY5, COP1 and DET1 were down-regulated, while MYB5b and Cry-2 were up-regulated. The results showed that, with the aging of tobacco leaves, the synthesis of carotenoids decreased and the degradation increased. The results from this study will facilitate in depth understanding of the molecular regulation of carotenoid metabolism during the maturation and senescence of tobacco leaves and promote genetic improvement of tobacco varieties targeting carotenoid content.