Isothiocyanates (ITCs), naturally occurring in abundance in cruciferous vegetables, are the most well-studied organosulfur compounds having an electrophilic reactivity. ITCs have been accepted as major ingredients of these vegetables that afford their health promoting potentials. ITCs are able to modulate protein functions related to drug-metabolizing enzymes, transporters, kinases and phosphatases, etc. One of the most important questions about the molecular basis for the health promoting effects of ITCs is how they modulate cellular target proteins. Although the molecular targets of ITCs remains to be validated, dietary modulation of the target proteins via covalent modification by ITCs should be one of the promising strategies for the protection of cells against oxidative and inflammatory damage. This review discusses the plausible target proteins of dietary ITCs with an emphasis on possible involvement of protein modification in their health promoting effects. The fundamental knowledge of ITCs is also included with consideration of the chemistry, intracellular behavior, and metabolism.