Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factor–α (TNFα), causing autocrine phosphorylation and activation of transforming growth factor–β–activated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38–induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress–induced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.