Here we evaluated the potential anti-pancreatic cancer activity by TIC10/ONC201, a first-in-class small-molecule inducer of tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL). The in vitro results showed that TIC10 induced potent cytotoxic and cytostatic activities in several human pancreatic cancer cell lines (Panc-1, Mia-PaCa2, AsPC-1 or L3.6). TIC10 activated both extrinsic (TRAIL-caspase-8-dependent) and endogenous/mitochondrial (caspase-9-dependent) apoptosis pathways in the pancreatic cancer cells. Molecularly, we showed that TIC10 inhibited Akt-Erk activation, yet induced TRAIL expression in pancreatic cancer cells. Significantly, TIC10, at a relatively low concentration, sensitized gemcitabine-induced growth inhibition and apoptosis against pancreatic cancer cells. Further, TIC10 and gemcitabine synergistically inhibited Panc-1 xenograft growth in SCID mice. The combination treatment also significantly improved mice survival. In addition, Akt-Erk in-activation and TRAIL/cleaved-caspase-8 induction were observed in TIC10-treated Panc-1 xenografts. Together, the preclinical results of the study demonstrate the potent anti-pancreatic cancer activity by TIC10, or with gemcitabine.