Artemisinin is a secondary metabolite extracted from Artemisia annua. As an effective antimalarial component certified by WHO, artemisinin has extensive economical values. Numerous studies about transcription factors positively regulating artemisinin biosynthesis have been published while negative regulators are rarely reported. In the present study, we identified AaMYB15 as the first R2R3-MYB that negatively regulates artemisinin biosynthesis in A. annua. Experimental evidences showed that AaMYB15 is a transcription factor within nucleus and predominantly expressed in glandular secretory trichomes (GSTs) in A. annua where artemisinin is synthesized and accumulated. The expression of AaMYB15 was induced by dark and JA treatment. Overexpression of AaMYB15 led to a significant decline in the expression levels of key enzyme genes ADS, CYP, DBR2, and ALDH1 and a significant decrease in the artemisinin contents of transgenic A. annua. AaMYB15 directly bound to the promoter of AaORA, a reported positive regulator of artemisinin biosynthesis in JA signaling pathway, to repress its transcriptional activity, thus downregulating the expression levels of downstream key enzyme genes and negatively regulating the artemisinin biosynthesis. Our study provides candidate gene for improvement of A. annua germplasm and new insights into the artemisinin biosynthesis regulation network mediated by light and JA.