The intracellular mechanisms of ischemic preconditioning (PC) in preventing lung dysfunction following transplantation, shock, and trauma remain poorly understood. Previously, we have shown that alveolar epithelial cells secrete calcitonin gene-related peptide (CGRP) under inflammatory stress. Using a hypoxia/reoxygenation (H/R) and PC model, we found that CGRP was also secreted from human type II alveolar epithelial cells (A549) after PC. The locally released CGRP interacted with its receptor on the membrane of A549 cells and elicited downstream signals mediating the PC effect, because hCGRP(8-37), a specific CGRP receptor antagonist, attenuated the protective effect of PC. Pre-inhibition of CGRP protein synthesis by small interfering RNA exacerbated (but overexpression of the CGRP gene ameliorated) H/R-induced cell death, which supports the autocrine effect of CGRP on A549 cells. Exogenous bioactive CGRP mimicked the beneficial effect of PC and up-regulated the expression of heat shock protein 70 (HSP70), which might act as the end effector to maintain cell viability. These effects were sensitive to hCGRP(8-37), calphostin C (a protein kinase C (PKC) inhibitor), and 5-hydroxydecanoic acid (a mitochondrial K(+)(ATP) channel blocker) but were insensitive to protein kinase A blockers. Moreover, CGRP induced the membrane translocation of PKCepsilon. PKCV1-2 (a cell-permeable inhibitory peptide of PKCepsilon) effectively abolished CGRP-induced HSP70 expression and cell protection. Therefore, PC induces CGRP secretion from human alveolar epithelial cells, and the locally released CGRP acts back on these cells, protecting them from H/R injury. The post-receptor signaling of CGRP is through PKCepsilon-dependent expression of HSP70.