Abstract Glucosinolates are specialized defensive metabolites characteristic of the Brassicales order. Among them, aliphatic and indolic glucosinolates (IGs) are usually highly abundant in species from the Brassicaceae family. The exceptions this trend are species representing a subclade of the Camelineae tribe, including Capsella and Camelina genera, which have reduced capacity to produce and metabolize IGs. Our study addresses the contribution of specific glucosinolate-related myeloblastosis (MYB) transcription factors to this unprecedented backward evolution of IG biosynthesis. To this end, we performed phylogenomic and functional studies of respective MYB proteins. The obtained results revealed weakened conservation of glucosinolate-related MYB transcription factors, including loss of functional MYB34 protein, in the investigated species. We showed that the introduction of functional MYB34 from Arabidopsis thaliana partially restores IG biosynthesis in Capsella rubella, indicating that the loss of this transcription factor contributes to the backward evolution of this metabolic pathway. Finally, we performed an analysis of the impact of particular myb mutations on the feedback loop in IG biosynthesis, which drives auxin overproduction, metabolic dysregulation and strong growth retardation caused by mutations in IG biosynthetic genes. This uncovered the unique function of MYB34 among IG-related MYBs in this feedback regulation and consequently in IG conservation in Brassicaceae plants.