Piperlongumine (PPLGM), a bioactive agent obtained from long pepper plants, possesses potent antitumor activity by inducing reactive oxygen species (ROS). However, the mechanisms for PPLGM’s antitumor actions are not well defined. We investigated PPLGM’s antitumor effects and molecular mechanisms against pancreatic and colon cancer, two of the leading causes of cancer death for both men and women in the U.S. We found that PPLGM activated a ROSmediated DNA damage pathway that lead to pancreatic cancer cell death in vitro. Further, mice treated with PPLGM showed reduced pancreatic tumor volume, which was associated with a decrease in tumor cell proliferation and enhanced oxidative stress levels. To elucidate the target pathways responsible for PPLGM-mediated cell death, RNA sequencing was performed. 684 genes were differentially expressed in pancreatic cancer cells treated with PPLGM compared to the control. Genes related to ER-stress and UPR pathways were activated in PPLGM-treated pancreatic cancer cells. To determine the therapeutic efficacy of PPLGM in combination with currently used chemotherapy in vivo, an orthotopic mouse model of pancreatic cancer was used. The combination of PPLGM with gemcitabine resulted in greater reduction of tumor weight and volume than either agent alone, and PPLGM-treated mouse tumors showed decreased expression of Ki-67, a proliferation marker. In vitro studies supported the in vivo results where the combination of PPLGM with gemcitabine and erlotinib significantly decreased pancreatic cancer cell viability and survival, and induced apoptosis compared to control cells or cells treated with the chemotherapeutic agents alone. PPLGM inhibited the growth of colorectal cancer cells to a greater degree than normal colon cells and activated p-ERK protein expression. The use of a MEK inhibitor attenuated the activation of p-ERK and partially blocked PPLGM-mediated cell death, indicating the involvement of the MEK/ERK pathway in colon cancer cell death. These results suggest PPLGM holds potential as a therapeutic agent to treat pancreatic and colon cancer in the clinics.