Glandular trichomes can secrete and store a large number of secondary metabolites in plants, some of which are of high medicinal and commercial value. For example, artemisinin, isolated from Artemisia annua L. plants, and its derivatives have great high medicinal value. Previous research indicated that artemisinin was synthesized in the glandular trichomes on the leaves of A. annua. It is an important study direction to improve artemisinin yield by promoting the initiation and development of glandular trichome. In this study, SQUAMOSA promoter-binding protein-like 9 (AaSPL9) was identified. In AaSPL9 overexpression transgenic plants, the glandular trichomes density was increased by 45-60 %, and the content of artemisinin was increased by 33-60 %, indicating that AaSPL9 positively regulate the glandular trichomes initiation. Yeast one-hybrid(Y1H), Dual-luciferase (Dual-Luc), Electrophoretic Mobility Shift Assay (EMSA) demonstrated that AaSPL9 activated the expression of AaHD1 by combining directly the GTAC-box of the AaHD1 promoter. Taken together, we identified AaSPL9, a positive transcription factor, regulating the glandular trichome initiation in A. annua, and revealed a novel molecular mechanism by which a SPL protein to promote glandular trichome initiation.