Isothiocyanates (ITCs) derived from cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit preventative effects against various types of cancers. Yet, the inhibitory effects of ITCs on C6 glioma cell invasion and migration have not been reported. Thus, we aimed to analyze ITC-regulated MMP-9 activation, a crucial enzyme of cancer metastasis that degrades the extracellular matrix, in C6 glioma cells to investigate the inhibitory effects on cancer invasion and migration by ITCs. In the present study, we found that ITCs specifically suppressed PMA-induced MMP-9 secretion and protein expression. The inhibitory effects of ITCs on PMA-induced MMP-9 expression were found to be associated with the inhibition of MMP-9 transcription levels through suppression of nuclear translocation of NF-κB and activator protein-1 (AP-1). It was also confirmed that ITCs decreased MMP-9-mediated signaling such as FAK and JNK, whereas they had no effect on the phosphorylation of ERK and p38. Moreover, wound-healing and Τranswell invasion assays showed that ITCs inhibited the migration and invasion of C6 glioma cells. These results suggest that ITCs could be potential agents for the prevention of C6 glioma cell migration and invasion by decreasing FAK/JNK-mediated MMP-9 expression.