BC-819 is a DNA plasmid that was developed to target the expression of diphtheria-toxin gene under the control of H19 regulatory sequences. BC-819 has the potential to treat pancreatic cancer that overexpresses the H19 gene. The objectives were to assess the safety, tolerability, pharmacokinetics and preliminary efficacy of BC-819 administered intratumorally in subjects with unresectable, locally advanced, non-metastatic pancreatic cancer. Nine patients with unresectable pancreatic adenocarcinoma were enrolled in an open-label, dose-escalation trial. Subjects were entered into one out of two cohorts with escalating doses of BC-819. Each cohort received 2 weeks of twice weekly intratumoral injection of BC-819 under computerized tomography (CT) (n = 3) or endoscopic ultrasound (EUS) (n = 6) guidance. Patients were assessed by CT or positron emission tomography (PET)/CT during week 4 for tumor response. The maximum tolerated dose of BC-819 was not reached in this study at the highest dose. Asymptomatic elevation of lipase, which was considered as an adverse event with dose-limiting toxicity, occurred in only one subject in the high-dose group and was resolved spontaneously. The tumors did not increase in size 4 weeks after initiating treatment. Two weeks after completing the treatment, the two subjects who went on to receive subsequent chemotherapy or chemoradiation therapy, pancreatic tumors were downstaged and considered surgically resectable. Remarkably, three of the six subjects in cohort no. 2 evaluated at month 3 had a partial response. BC-819 can be safely administered intratumorally via EUS- or CT-guided injection at a dose of at least 8 mg per injection weekly twice. BC-819 given locally in combination with systemic chemotherapy may provide an additional therapeutic benefit for the treatment of pancreatic cancer.