Resistance to drug treatments underlies the high lethality of pancreatic ductal adenocarcinoma. Along with others, we have recently identified that proteasome inhibition is a promising therapeutic option in this highly refractory disease. The pleiotropic effects of proteasome inhibition include the activation of apoptotic signaling pathways and also antiapoptotic signaling pathways such as EGFR, AKT and the MAP kinases that reduce the apoptotic potential of this class of drug. In this study, we sought to determine the mechanism behind the activation of EGFR in response to proteasome inhibition in pancreatic cancer cells. We found that the second-generation proteasome inhibitor NPI-0052 induced the mRNA transcription of several EGFR family ligands (EGF, HB-EGF and epiregulin), however only increases in HB-EGF were detected at the protein level. Using both pharmacological inhibitors and lentiviral-mediated shRNA knockdown of EGFR ligand expression, we discovered that ligand cleavage by MMP/ADAMs and HB-EGF expression is required for activation of EGFR in response to proteasome inhibition. Furthermore, we discover that induction of HB-EGF is dependent on reactive oxygen species and p38-MAPK signaling but not ERK and that the transcription factor SP-1 is involved in NPI-0052-induced HB-EGF transcription. Together, these results indicate that stress signaling leading to induction of HB-EGF expression and increases in MMP/ADAM-dependent HB-EGF cleavage are responsible for proteasome inhibitor-induced activation of EGFR in pancreatic cancer cells.