Sesquiterpene lactone artemisinin is one of the main agents used to treat malaria. Artemisinin is produced in the glandular secretory trichomes (GSTs) of Artemisia annua. The plant hormones and metabolic pathways regulate the artemisinin content of A. annua. It was possible to examine the functions of auxin, an important plant hormone, in the development of GSTs in A. annua by enhancing the expression of iaaM, which encodes a tryptophan monooxygenase involved in the biosynthesis of auxin. Additionally, the effects of co-overexpression of aldehyde dehydrogenase 1 (ALDH1), P450 monooxygenase (CYP71AV1), and iaaM on the density, length, and width of GSTs and the contents of artemisinin were further investigated. Results indicated that overexpression of iaaM might increase the density, length, and width of GSTs by enhancing auxin biosynthesis. This study also proved the key regulatory role of ALDH1 in the biosynthesis of artemisinin. Moreover, co-overexpression of ALDH1, CYP71AV1, and iaaM successfully increased the density, length, and width of GSTs and improved the artemisinin content in A. annua. Therefore, we established a theoretical basis for modifying artemisinin accumulation in this study by regulating the expression of auxin and artemisinin synthesis-related genes using a metabolic engineering method.