Abstract: The action mechanism of forchlorfenuron (CPPU, a benzylurea cytokinin) on the quality of upper flue-cured leaves was investigated through a field experiment in which different mass concentrations (10, 20, and 50 mg/L) of CPPU were sprayed and the results were compared with water spray as a control. The effects of CPPU on agronomic traits, chemical composition, appearance, and sensory quality of Yunyan 87 and Yunyan 97 tobacco cultivars were investigated. The results showed that the effects of different concentrations of CPPU on the two cultivars were different from the control. In terms of overall efficacy, both Yunyan 87 and Yunyan 97 showed the best effect after being sprayed with a concentration of 20 mg/L CPPU. The upper leaves of Yunyan 87 sprayed with CPPU at a mass fraction of 20 mg/L were used as research materials, and the results on the leaf's cell structure, gene expression, and endogenous hormone content (mass fraction) analysis showed that the upper and lower epithelial cells of Yunyan 87 leaves increased by 17.21%-53.94% and 71.05%-75.79%, respectively, as compared with the control. In addition, leaf thickness increased from 32.41% to 44.31%. Furthermore, there was an upregulation in the relative expression level of NtTOR1 gene, which is responsible for regulating cell volume. The application of CPPU resulted in an increase in the levels of cytokinins, auxins, and gibberellins, thereby inducing an upregulation of the relative expression of key genes involved in cytokinin signal transduction (NtABRB-1), auxin signal transduction (NtABP1), and gibberellin metabolism (NtGRAS). Notably, this effect was most pronounced 12 days after the CPPU treatment. Therefore, exogenous CPPU can upregulate the expression of signal transduction-related genes by increasing the contents (mass fractions) of cytokinins, auxins, and gibberellins in leaf cells, thereby improving tobacco quality.