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Arbuscular mycorrhizal (AM) is the most widespread form of plant symbiosis and has been found to enhance photosynthesis rate, biomass accumulation, pathogen defense, and tolerance to heavy metals and cold stress. In addition, it affects the production of plant secondary metabolites, one of which is camptothecin, an anti-cancer compound used in clinical practice. Camptothecin is a water-insoluble cytotoxic monoterpene derived from indole alkaloid and was initially isolated from the Chinese tree Camptotheca acuminate (Nyssaceae). It has gained great attention for its remarkable inhibitory activity against tumor cells. Based on our previous study on the correlation between the formation of mycorrhiza and camptothecin content in mycorrhizal C. acuminata seedlings, the effect of co-cultivation time on camptothecin accumulation in C. acuminata seedlings after inoculation with Acaulospora mellea and Glomus intraradices was investigated in the present study. Seeds of C. acuminata were sterilized and sown in sterilized matrix (a mixture of soil and sand) in the