Aliphatic glucosinolates (AGSLs) constitute an important part of sulfur-containing secondary metabolites in Arabidopsis. Biosynthesis of AGSLs is positively regulated by transcription factors MYB28, MYB29, and MYB76. Compared to plants grown under full S conditions (1,500 μM sulfate), in wild type Arabidopsis, the AGSL content was reduced to nearly 70 % when grown under 1/10 S conditions (150 μM sulfate) and nearly disappeared in plants grown under 0 S conditions. The expression of MYB29 and MYB76 was positively correlated with sulfur concentration, whereas the expression of MYB28 was slightly elevated in lines grown under 1/10 S conditions, and maintained at basal levels under 0 S conditions. To eliminate the effects of MYB interaction, transgenic lines in which one of these three MYB genes was expressed in the myb28myb29 background, were subjected to sulfur deficiency. In the absence of MYB29 and MYB76, an apparent increase of MYB28 expression level in Pro MYB28 :MYB28 lines was detected when grown under 1/10 S conditions. Altering the regulation of MYB genes allows the plants to allocate the limited sulfur resources between primary and secondary metabolism under sulfur deficiency.