Glucosinolates (GSLs) are a group of plant secondary metabolites that have repellent activity against herbivore insects and pathogens, and anti-carcinogenic activity in humans. They are produced in plants of the Brassicaceae and other related families. Biosynthesis of GSLs from precursor amino acids takes place in two subcellular compartments; amino acid biosynthesis and side chain elongation occur mainly in the chloroplast, whereas the following core structure synthesis takes place in the cytosol. Although the genes encoding biosynthetic enzymes of GSLs are well known in Arabidopsis thaliana, the transporter genes responsible for translocation of biosynthetic intermediates between the chloroplast and cytosol are as yet unidentified. In this study, we identified the bile acid:sodium symporter family protein 5 (BASS5) gene in Arabidopsis as a candidate transporter gene involved in methionine-derived GSL (Met-GSL) biosynthesis by means of transcriptome co-expression analysis. Knocking out BASS5 resulted in a decrease of Met-GSLs and concomitant increase of methionine. A transient assay using fluorescence fusion proteins indicated a chloroplastic localization of BASS5. These results supported the idea that BASS5 plays a role in translocation across the chloroplast membranes of the biosynthetic intermediates of Met-GSLs.