Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCδ to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCδ inhibitor rottlerin and a PKCδ kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCδ in the apoptosis induced by SVNI we used a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). PKC δ5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCδ. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCδ5 but did not induce cleavage of PKCδ or PKC δ5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCδ exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCδ on specific tyrosines is required for this function.