Protein kinase C-δ (PKCδ) plays an important role in DNA damage-induced apoptosis. We have previously shown that the PKCδ inhibitor rottlerin protects against cisplatin-induced apoptosis acting upstream of caspase-9. In the present study, we have investigated if rottlerin regulates caspase-2 activation. Knockdown of caspase-2 by siRNA inhibited processing of apical caspase-9 and caspase-8, whereas depletion of caspase-9 had little effect on caspase-2 processing. Rottlerin inhibited activation and processing of caspase-9 and caspase-8 and cleavage of poly(ADP)ribose polymerase. We made a novel observation that rottlerin induced down-regulation of caspase-2 but not of caspase-3, caspase-7, caspase-8, or caspase-9. Pharmacologic inhibitors of PKC, such as Gö 6983 and bisindolylmaleimide, or depletion of PKCδ by siRNA had no effect on the down-regulation of caspase-2 by rottlerin. The proteasome inhibitor MG132 reversed caspase-2 down-regulation by rottlerin, whereas calpain inhibitor had no effect. These results suggest that rottlerin induces down-regulation of caspase-2 via PKCδ-independent but ubiquitin proteasome-mediated pathway. Furthermore, down-regulation of caspase-2 by rottlerin can explain its antiapoptotic function during DNA damage-induced apoptosis.