Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKCδ is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKCδ by RNAi (sh-PKCδ) or expression of the kinase-dead (Lys376Arg) mutant of PKCδ (PKCδKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (γ-H2AX), a hallmark for DNA damage, was altered by sh-PKCδ, and sh-PKCδ completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKCδ-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKCδ and caspase-3, suggesting that PKCδKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKCδ-dependent and -independent manners. In summary, these findings suggest that the activation of a PKCδ-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment.