Protein kinase D (PKD), a downstream effector of protein kinase C (PKC), is implicated in suppression of the c-Jun N-terminal kinase (JNK) signaling pathway, however, its mechanism of action is unclear. Transphosphorylation of the PKD activation loop at serines 744/748 by a PKC mediated signal transduction pathway enhances its catalytic activity. Here we show that PKD activation loop phosphorylation at serines 744/748 via PKC, or mutation of these serines to glutamic acid (PKD-S744/748E) also results in complex formation with JNK, indicating that suppression of JNK signaling by PKD involves a direct interaction with JNK. Because catalytically active PKD associates with JNK we determined whether it could phosphorylate the c-Jun N-terminus as a potential mechanism by which it suppresses c-Jun Ser 63 phosphorylation when it complexes with JNK. Purified human PKD and either wild-type PKD from phorbol 12, 13-dibutyrate (PDB)-stimulated cells or unstimulated constitutively active PKD (PKD-S744/748E), phosphorylated the c-Jun N-terminus between amino acids 1-89 at sites distinct from those phosphorylated by JNK. These results demonstrate, for the first time, phosphorylation dependent association of PKD with another signaling molecule and reveal a potential mechanism by which PKD could modulate the ability of JNK to phosphorylate c-Jun by phosphorylating alternative sites in the c-Jun N-terminus when it is complexed with JNK.