Camptotheca acuminata is the major plant for the production of camptothecin (CPT), an important anticancer drug used for the treatment of various cancers throughout the world. The low accumulation of CPT in plants limited its supply in the market and led to urgent need of promoting its accumulation by means of plant metabolic engineering, which relied on deep understanding of CPT biosynthesis pathway. However, missing of most of pathway genes restricted the attempts for regulating CPT biosynthesis. To unveil the CPT biosynthesis pathway, many efforts have been done for pathway gene identification and the application of large-scale RNA-sequencing technique accelerated screening of candidate genes which could be involved in CPT biosynthesis. To identify the function of these candidate genes in planta, it needs to develop an effective approach, such as virus-induced gene silencing (VIGS) in C. acuminata. In this work, a Tobacco Rattle Virus-based VIGS method was developed and the application of this method successfully silenced the expression of four known genes involved in the early steps of CPT biosynthesis and resulted in clear decrease of CPT and 10-hydroxycamptothecin (10-HCPT) accumulation. This VIGS approach could be further applied to functional identification of candidate genes to elucidate CPT biosynthesis in C. acuminata.