Background: Despite significant efforts, the artemisinin-based drugs are still very expensive due to the limited production of this metabolite within wild Artemisia spp . plants. Therefore, the current work set out to evaluate the effect of chitosan nanoparticles, as a novel elicitor to characterize the expression of genes functioning in artemisinin synthesis pathway using a comparative experimental investigation. Methods: The suspensioncultures of A. anuua were exposed to 5, 10, 15 mg/L of chitosan nanoparticles (during 8, 24, 48 and 72 h upon treatment). The expression of DBR2, SQS, CYp , ADS, CPR and ALDH genes were quantified by qRT-PCR technique. Results: Chitosan nanoparticles were effective in inducing artemisinin production at 15 mg/L after 8 h, and 5 and 10 mg/L after 72 h of elicitation, in which all the ADS, CYp , CPR, DBR2 and ALDH genes were upregulated except SQS. Conclusion: The treatment of 5 mg/L after 72 h, when cells entered the stationary and then death phases, is recommended because it seems chitosan nanoparticles require more time to up-regulate the ADS, CYP and ALDH genes and thereby probably enhance the artemisinin content. The results suggest that chitosan nanoparticles can be used as a novel effective elicitor for artemisinin production.