CUB (C1r/C1s, urchin embryonic growth factor, BMP1) domain-containing protein 1 (CDCP1) has been implicated in promoting metastasis of cancer cells through several mechanisms, including the inhibition of anoikis, which is cell death triggered by the loss of extracellular matrix interactions. However, the mechanism inhibiting cell death regulated by CDCP1 remains elusive. Inhibition of CDCP1 expression using small interfering RNA (siRNA) induced the cell death of suspended cancer cells without cleaving caspase-3, a marker of apoptosis; cell death was not inhibited by a general caspase inhibitor, suggesting that the loss of CDCP1 induces caspase-independent cell death. In contrast, knockdown of CDCP1 as well as protein kinase Cδ (PKCδ), a downstream effector of CDCP1, in a suspension culture of lung cancer cells resulted in marked induction of membranous microtubule-associated protein 1 light chain 3 (LC3)-II protein, a hallmark of autophagy, and caused the formation of an autophagosome structure visualized using green fluorescent protein-tagged LC3-II. Expression and phosphorylation of exogenous CDCP1 by Fyn kinase reduced the formation of autophagosomes and inhibited phosphorylation of CDCP1 by PP2, a Src kinase inhibitor or inhibited PKCδ by rottlerin, stimulating autophagosome formation. Moreover, death of suspended lung cancer cells induced by CDCP1 siRNA or by PKCδ siRNA was reduced by the autophagy inhibitor 3-methyladenine. These results indicate that CDCP1-PKCδ signaling plays a critical role in inhibiting autophagy, which is responsible for anoikis resistance of lung cancer cells.