The interplay of plant hormones and glucose (Glu) in regulating glucosinolate accumulation in Arabidopsis thaliana was investigated in this study. Glucose-induced glucosinolate biosynthesis was enhanced significantly by the addition of jasmonic acid (JA), whereas the synergistic effect of salicylic acid (SA) and Glu was less obvious. The enhanced glucosinolate accumulation is associated with elevated expression of genes in glucosinolate biosynthetic pathway, as well as the transcription factors involved in their regulation, such as MYB28, MYB29, MYB34, and MYB122. The induction of indolic and aliphatic glucosinolates after treatment with JA and Glu in JA-insensitive mutants, coi1, jar1, and jin1, was compromised. Moreover, the effect of JA and Glu on glucosinolate contents was dramatically reduced in Glu-insensitive mutants, rgs1-2 and abi5-7. These results indicate a crosstalk between JA and Glu signalling in the regulation of glucosinolate biosynthesis. JA signalling, RGS1 (the putative membrane receptor of Glu signalling), and ABI5, are involved in the synergistic effect of JA and Glu on glucosinolate accumulation.