Circular RNAs (circRNAs), as one of important regulators, play important roles in various plant biol. processes, including stress responses, and variations in gene expression patterns.However, the regulatory role of circRNAs in secondary metabolite biosynthesis is largely unexplored.In this study, by circRNA-sequencing of Nicotiana tabacum, of which nicotine accounts for 90 % of total alkaloids, a total of 36,596 circRNAs were identified for five tissues across three developmental periods.Numerous circRNAs exhibited tissue-specific expression patterns compared to their host genes.Based on genomic information of circRNAs and comparing between topping treatments, a subnetwork was constructed for nicotine biosynthesis.This network revealed that 52 circRNAs could target known nicotine biosynthesis genes and 60 circRNAs could target known jasmonate acid (JA) biosynthesis genes.Furthermore, 154 differentially expressed circRNAs were identified as potential novel regulators of nicotine biosynthesis.Overexpression of circRNA NRC1 significantly elevated the accumulation of nicotine and JAs.Overall, these findings demonstrate the critical role of circRNAs in nicotine biosynthesis.