Background: Treatment of Pancreatic Cancer (PC) is challenging due to its aggressiveness and acquired resistance to conventional chemotherapy and radiotherapy. Therefore, the discovery of new therapeutic agents and strategies is essential. Juglone, a naphthoquinone, is a secondary metabolite produced naturally in walnut-type trees having allelopathic features in its native environment. Juglone was shown to prevent cell proliferation and induce ROS-mediated mitochondrial apoptosis. Ascorbate with both antioxidant and oxidant features, shows selective cytotoxicity in cancer cells. Methods and results: In this study, we evaluated the anticancer effects of Juglone in combination with ascorbate in PANC-1 and BxPC-3 PC cells. The MTT assay was used to determine the IC50 dose of Juglone with 1 mM NaAscorbate (Jug-NaAsc). Subsequently, the cells were treated with 5, 10, 15 and 20 µM Jug-NaAsc for 24 h. Apoptotic effects were evaluated by analyzing the following genes using qPCR; proapoptotic Bax, antiapoptotic Bcl-2 related to the mitochondrial apoptotic pathway and apoptosis inhibitor Birc5 (Survivin). Immunofluorescence analysis was performed using Annexin V-FITC in PC cells. As an antioxidant enzyme, Trx2 protein levels were determined by a commercial ELISA test kit. Jug-NaAsc treatment decreased the expressions of antiapoptotic genes Bcl-2 and Birc5 while the apoptotic gene Bax expression increased at all doses. Additionally, a dose-dependently increase of apoptosis according to immunofluorescence analysis and the decreases of Trx2 enzyme levels at all treatments in both cell lines supported gene expression results. Conclusion: Our results suggest that Juglone is a potential anticancer agent especially when combined with ascorbate.