Although protein kinase Cδ (PKCδ) has been suggested in the negative control of the cell cycle machinery in many types of cancer cells, its underlying mechanisms are partly understood. Here we report that the expression of apoptosis signal-regulating kinase1 (ASK1) is inducible in a PKCδ-dependent manner, and contributes to phorbol ester-induced cell cycle arrest through persistent JNK activation in breast cancer epithelial cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) gradually up-regulated the expression of ASK1 mRNA and protein, and subsequently enhanced its catalytic activity in MCF-7 cells. Importantly, such PMA-induced ASK1 expression was completely abolished by pretreatment of rottlerin, a specific PKCδ inhibitor or by knocking down the expression of PKCδ, while ectopic expression of a constitutively active form of PKCδ strongly up-regulated ASK1 expression. We also found that the persistent activation of mitogen-activated protein kinase, JNK in response to PMA was greatly attenuated by RNA interference-mediated knockdown of ASK1. Taken together, these results suggest that inducible expression of ASK1 by PKCδ contributes to the G1 arrest by enhancing persistent JNK signaling activation which represents a novel alternative mechanism of PKCδ-dependent cell cycle arrest and limiting proliferation of breast cancer epithelial cells.