Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies because it is often diagnosed at a late disease stage and has a poor response rate to currently available treatments. Therefore, it is critical to develop new therapeutic approaches that will enhance the efficacy and reduce the toxicity of currently used therapies. Here we aimed to evaluate the therapeutic potential and mechanisms of action for piperlongumine (PL), an alkaloid from long pepper, in PDAC models. We postulated that PL causes PDAC cell death through oxidative stress and complements the therapeutic efficacy of chemotherapeutic agents in PDAC cells. First, we determined that PL is one of the most abundant alkaloids with antitumor properties in the long pepper plant. We also showed PL in combination with gemcitabine, a chemotherapy agent used to treat advanced pancreatic cancer, reduced tumor weight and volume compared to vehicle-control and individual treatments. Further, biochemical analysis, including RNA sequencing and immunohistochemistry, suggested that the antitumor activity of PL was associated with decreased cell proliferation, induction of cell cycle arrest, and oxidative stress-induced cell death. Moreover, we identified that c-Jun N-terminal kinase (JNK) inhibition blocks PL-induced cell death, translocation of Nrf2, and transcriptional activation of HMOX1 in PDAC. Finally, high-throughput drug and CRISPR screenings identified potential targets that could be used in combination with PL to treat PDAC cells. Collectively, our data suggests that cell cycle regulators in combination with PL might be an effective approach to combat pancreatic cancer.