Cardioprotective activity of sulforaphane (SF) has been predominantly associated with its ability to induce phase II detoxification enzymes. In the present study, novel targets of SF were identified and characterized using a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to produce protein profiles of rat cardiomyocytes treated with 5 µM SF for 1-48 h. Gel comparisons showed the regulation of nearly 60 proteins. In particular we observed a strong modulation of macrophage migration inhibitory factor, lactoylglutathione lyase, elfina and HSP60. Moreover, proteins were analyzed using western blotting. Results suggested that SF cardioprotection is a complex mechanism involving not only the induction of phase II enzymes1, but also unexpected proteins with antiapoptotic role2 or acting as adapters between kinases and cytoskeleton3. This study contributed to clarify the mechanisms behind SF cardioprotection.