Abstract: To investigate the correlation between maturity and stiffness of upper leaves of flue-cured tobacco, the upper leaves of Yunyan 87 were selected as the test material and classified into mature tobacco leaves (JY) and ripe tobacco leaves (CK) according to their maturity. The relationship between maturity and stiffness of tobacco leaves was analyzed at the cell biological and molecular levels through dark box experiments, transmission electron microscopy observation and fluorogenic quantitative PCR techniques. The results showed that: 1) The easiness and tolerance of flue-curing of mature tobacco leaves were worse than those of ripe tobacco leaves. 2) GO and KEGG enrichment analyses of the transcriptome differential genes of tobacco leaves at two maturity levels revealed that differential genes were more enriched in starch and sucrose metabolism, indicating that the formation of stiff tobacco leaves was closely related to starch and sucrose metabolism. 3) Cell ultrastructure observation showed that the starch granules and osmiophilic granules were larger in the cells of mature tobacco leaves and the starch granules were hard to degrade during curing process. 4) At the yellowing stage, the hardness and starch content of mature tobacco leaves were higher than those of ripe tobacco leaves, while the respiratory intensity and total sugar content of mature tobacco leaves were significantly lower than those of ripe tobacco leaves. 5) The analysis of conventional chemical components in cured tobacco leaves showed that the starch content of mature tobacco leaves was significantly higher than that in ripe tobacco leaves, while the contents of total sugar, total alkaloid, and total petroleum ether extract were significantly lower than those in ripe mature leaves. 6) Compared with mature tobacco leaves, the relative expressions of NtAGPS, NtGBSS, NtSS and NtSBE, which were key genes for starch synthesis in ripe tobacco leaves, were significantly reduced, while the relative expressions of starch hydrolase genes NtAMML-α and NtAMML-β were significantly upregulated. 7) Compared with mature tobacco leaves, the relative expression levels of the key genes in glucose metabolism pathway NtSPS, NtSUS and NtINV were significantly reduced in ripe tobacco leaves, while the relative expression of NtSPP showed no significant change. The starch content in mature tobacco leaves was high, and was difficult to degrade during curing, indicating that insufficient starch metabolism in the upper leaves of flue-cured tobacco with low maturity was an important reason for the high proportion of stiff tobacco leaves of cured tobacco.