Cellular stress responses are evolutionarily-conserved defense mechanisms used by the cell to upregulate cytoprotective genes and protect against harmful damage. If resolution fails, the cell will succumb to one of several programmed cell death pathways. Plasminogen activator inhibitor type 2 (PAI-2) is a multifunctional serine protease inhibitor (serpin) that is produced in response to stressful stimuli. Deregulation of PAI-2 gene expression is implicated in cancer as well as a host of other inflammatory diseases. Thus, we investigated molecular mechanisms that regulate inflammatory stress-associated PAI-2 gene expression and a mechanism for its cytoprotective activity. Our results demonstrate that the PAI-2 proximal promoter confers the greatest response to lipopolysaccharide (LPS), a hallmark inflammatory agent. Further, we found that the PAI-2 proximal promoter is critically dependent on a CCAAT enhancer binding (C/EBP) element which binds the transcription factor C/EBP-β. Importantly, both constitutive and LPS-induced PAI-2 gene expression was severely abrogated in C/EBP-β-null mouse embryonic fibroblasts and primary C/EBP-β-deficient peritoneal macrophages. The major role of intracellular PAI-2 is cytoprotection, which is dependent on its serpin inhibitory activity; however an intracellular protease target remains unidentified. Interestingly, recent evidence from our lab suggests that calpain may be a candidate protease target for intracellular PAI-2. Calpain activity reportedly plays a role in cell death; therefore we tested the hypothesis that PAI-2 mediates cytoprotection by acting as an intracellular inhibitor of calpain activity. We found that recombinant PAI-2 was able to inhibit recombinant calpain activity in vitro. Wildtype (WT) PAI-2, stably expressed in human ovarian cancer cell lines, protected against cell death induced by calcimycin and thapsigargin - two activators of calpain activity; whereas expression of non-inhibitory serpin mutants of PAI-2 abrogated this effect. Surprisingly, we did not detect cleavage of common apoptotic markers which are reported calpain substrates in our cellular model. However, evidence suggests that PAI-2 may modulate an autophagic response because we found that cells expressing PAI-2 showed increased expression of microtubule-associated light chain 3 B (LC3-II) following treatment, as well as increased susceptibility to Chloroquine-induced cell death. Taken together, these data provide new insight into the regulation of PAI-2 gene expression and its cytoprotective activity.