Abstract Rejuvenation refers to the transition from an adult state to a juvenile state. Trunk truncation at the base of the tree can result in tree rejuvenation. However, little is known about the association of rejuvenation with leaf biomass and flavonoid accumulation. The results of this study showed that, compared with control leaves, leaves of renewed Ginkgo biloba shoots were larger, thicker, and more lobed and had higher fresh/dry weights and chlorophyll contents. The leaf biomass per hectare of rejuvenated trees was twofold higher than that of the untruncated controls. Moreover, we observed a marked increase in the accumulation of flavonol glycosides via metabolomic analysis and detected upregulated expression of genes involved in flavonoid biosynthesis, including CHS, FLS, F3’H, DFR, and LAR. Overexpression of GbCHS in ginkgo calli confirmed that GbCHS plays an important role in flavonoid biosynthesis. Interestingly, the contents of gibberellins significantly increased in the rejuvenated leaves. Moreover, exogenous gibberellin treatment significantly increased GbCHS expression and flavonoid contents. Our findings show that truncation can stimulate tree rejuvenation by altering hormone levels, representing an effective and feasible approach for enhancing the biomass and flavonoid content of G. biloba leaves.