Glucosinolates (GSL) and their derivatives are well known for the characteristic roles they play in plant defense as signaling molecules and as bioactive compounds for human health. More than 130 GSLs have been reported so far, and most of them belong to the Brassicaceae family. Several enzymes and transcription factors involved in the GSL biosynthesis have been studied in the model plant, Arabidopsis, and in a few other Brassica crop species. Recent studies in GSL research have defined the regulation, distribution, and degradation of GSL biosynthetic pathways; however, the underlying mechanism behind transportation of GSLs in plants is still largely unknown. This review highlights the recent advances in the metabolic engineering of GSLs in plants and discusses their potential applications.