Isothiocyanates (ITCs) are phytochemicals highly expressed in cruciferous vegetables and these compounds are associated with the decreased incidence of cancers in populations consuming high levels of cruciferous vegetables. Several individual ITCs including phenethyl isothiocyanate (PEITC) inhibit tumor growth and angiogenesis and their anticancer activity has been linked to inhibition of cancer cell growth, survival and inflammation (NFB). It has also been demonstrated that PEITC induces reactive oxygen species (ROS) and that ROS is largely responsible for PEITC-induced cell death. To confirm PEITC-induced cancer cell death we have investigated the mechanism of action of PEITC in pancreatic cancer cell lines and PEITC induces ROS and inhibits growth and induces apoptosis (PARP cleavage). In addition, PEITC downregulates expression of several gene products including vascular endothelial growth factor (VEGF), cyclin D1 (CD1), Bcl2 and survivin and these have previously been reported in other studies. However, since these gene products are all regulated by specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, which are overexpressed in cancer cells and tumors, we investigated the effects of PEITC on Sp proteins and observed that PEITC decreased expression of Sp1, Sp3 and Sp4 in pancreatic cancer cells. These results demonstrate for the first time that an important underlying mechanism of action of ITCs likely involves targeting Sp transcription factors through an ROS-mediated mechanism and the pathways required for ITC-induced Sp downregulation were investigated and the results are presented in this paper.