Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has raised attention as a novel anticancer therapeutic as it induces apoptosis preferentially in tumor cells. However, first-generation TRAIL-receptor agonists (TRAs), comprising recombinant TRAIL and agonistic receptor-specific antibodies, have not demonstrated anticancer activity in clinical studies. In fact, cancer cells are often resistant to conventional TRAs. Therefore, in addition to TRAIL-sensitizing strategies, next-generation TRAs with superior apoptotic activity are warranted. APG350 is a novel, highly potent TRAIL-receptor agonist with a hexavalent binding mode allowing the clustering of six TRAIL-receptors per drug molecule. Here we report on preclinical in vitro and in vivo studies testing the activity of APG350 on pancreatic ductal adenocarcinoma (PDAC) cells. We found that APG350 potently induced apoptosis of Colo357, PancTuI and Panc89 cells in vitro. In addition, APG350 treatment activated non-canonical TRAIL signaling pathways (MAPK, p38, JNK, ERK1/ERK2 and NF-κB) and induced the secretion of IL-8. Stable overexpression of Bcl-xL inhibited APG350-induced cell death and augmented activation of non-canonical pathways. Intriguingly, pre-treatment of Bcl-xL-overexpressing cells with the BH3-mimic Navitoclax restored their sensitivity to APG350. To study the effects of APG350 on PDAC cells in vivo, we applied two different orthotopic xenotransplantation mouse models, with and without primary tumor resection, representing adjuvant and palliative treatment regimes, respectively. APG350 treatment of established tumors (palliative treatment) significantly reduced tumor burden. These effects, however, were not seen in tumors with enforced overexpression of Bcl-xL. Upon primary tumor resection and subsequent APG350 treatment (adjuvant therapy), APG350 limited recurrent tumor growth and metastases. Importantly, therapeutic efficacy of APG350 treatment was more effective compared with treatment with soluble TRAIL in both models. In conclusion, APG350 represents a promising next-generation TRA for the treatment of PDAC. Moreover, our results suggest that combining APG350 with Navitoclax might be a succesfull strategy for cancers harboring mitochondrial apoptosis resistance.