<p><i>Ginkgo biloba</i> L. callus cultures (GbCCs) are emerging as a promising alternative to leaves for the in vitro production of bioactive secondary metabolites (flavonoids and terpenoids). This study investigated the influence of LED lighting on cell growth and the biosynthesis of flavonoids and terpenoids in GbCCs for the first time. Results showed that different colors of LED lights exerted distinct regulatory effects on cell growth and the accumulation of chlorophylls, flavonoids, and terpenoids in GbCCs. Compared with the dark control, all LED lights inhibited terpenoid accumulation but promoted flavonoid production, especially blue light. The total yield of nine flavonoids (1.90 ± 0.29&#xa0;mg/g DW) significantly improved (15.71-fold increase) in GbCCs exposed to blue light (3&#xa0;W/m²) for 40 days. Blue light significantly promoted the elongation of callus cells and induced antioxidant responses in GbCCs. Moreover, blue light activated the expression of the photoreceptor gene (<i>CRY1</i>) and <i>HY5</i>, thereby upregulating the expression of <i>MYB</i> and several enzyme genes (e.g., <i>FLS</i>, <i>F3’H</i> and <i>F3’5&#xa0;H</i>) responsible for flavonoid biosynthesis in GbCCs. The activation of <i>miR163a</i>/<i>b</i> by <i>HY5</i> repressed the expression of <i>AP2</i> and <i>bHLH</i>, consequently downregulating the expression of enzyme genes in the terpenoid biosynthesis pathway. Furthermore, the transcriptional activation of <i>miR163a</i>/<i>b</i> was also associated with the elongation of callus cells. Overall, the supplemental blue LED lighting represents a promising strategy for boosting the accumulation of bioactive flavonoids in GbCCs. These findings also provide valuable insight into the photo-regulatory mechanisms underlying secondary metabolism in <i>G. biloba</i>.</p> Graphical Abstract <p></p>