Isothiocyanates (ITCs) are considered major components of cruciferous vegetables that provide their disease-preventing and health-promoting effects. ITCs have the potential to modulate protein functions through their electrophilic characteristics, which might be involved in the molecular mechanisms of cytoprotection against oxidative stress as well as exogenous toxins. On the other hand, the chemical instability and metabolic susceptibility of ITCs might play a negative regulatory role in their biological activities. In this chapter, the fundamental knowledge of ITCs, such as their chemistry and metabolism, is introduced. We discuss the target proteins of ITCs and their role in health-promoting effects. This chapter also shows that improving the stability and the presence of coexisting substances are potential strategies to potentiate the biological activity of ITCs.