Artemisinin is a sesquiterpene lactone synthesized by the Chinese medicinal plant Artemisia annua L., and is the principal component used for the treatment of malaria. Recent studies have demonstrated the potential role of B-box (BBX) transcription factors in regulating plant secondary metabolism. However, the roles and functions of BBX members in the biosynthesis of artemisinin in A. annua have not been reported yet. In this study, a total of 27 BBX members (AaBBXs) were identified and classified into five sub-groups (I-V). The AaBBXs in each sub-group shared similar gene structures, conserved domains and motifs. Additionally, multiple cis-acting elements which are responsible for light, hormones, and stress signals were found in the promoter regions of AaBBX members. Based on transcriptome data, 27 AaBBXs showed constitutive expression in different organs/tissues, while several BBX genes exhibited higher expression levels in glandular secretory trichomes (GSTs), the site of artemisinin biosynthesis. To explore the roles of AaBBX members in regulating the artemisinin biosynthesis, 7 AaBBXs (AaBBX5/6/8/15/22/23/24) were selected for further investigation. The expressions of all 7 selected AaBBXs were strongly induced by MeJA and ABA, and they were all found to be nuclear localized. Additionally, all seven proteins were found to have the ability to physically interact with AaJAZ8 in vitro. Dual-luciferase assays revealed that AaBBX5/6/8/15/22/23 can activate the promoter activity of the artemisinin biosynthetic pathway genes such as AaADS, AaCYP71AV1, AaDBR2 and AaALDH1 at different degrees. AaBBX22 showed highest expression level in A. annua GSTs, and can significantly activate the promoters of all four artemisinin biosynthetic pathway genes. Furthermore, transgenic assay results indicated that AaBBX22 acted as a positive regulator for the biosynthesis of artemisinin in A. annua. Taken together, this study provided a comprehensive genome-wide characterization of the BBX family and identified several significant BBX genes, including AaBBX22, which paves the way for further investigation into the regulatory role of the BBX gene family in artemisinin biosynthesis.